iRNA Agents Targeting VEGF

ABSTRACT

The features of the present invention relate to compounds, compositions and methods useful for modulating the expression of vascular endothelial growth factor (VEGF), such as by the mechanism of RNA interference (RNAi). The compounds and compositions include iRNA agents that can be unmodified or chemically-modified.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/620,212 (pending), filed Sep. 14, 2012, which is a divisional of U.S.application Ser. No. 13/043,228, filed Mar. 8, 2011, now U.S. Pat. No.8,293,719, which is a divisional of U.S. application Ser. No.11/078,073, filed Mar. 11, 2005, now U.S. Pat. No. 7,947,659. U.S.application Ser. No. 13/043,228 is also a divisional of U.S. applicationSer. No. 11/340,080, filed Jan. 25, 2006, now U.S. Pat. No. 7,919,473,which is a continuation-in-part application of U.S. application Ser. No.11/078,073. U.S. application Ser. No. 11/078,073, filed Mar. 11, 2005,now U.S. Pat. No. 7,947,659, claims the benefit of U.S. ProvisionalApplication No. 60/552,620, filed Mar. 12, 2004; U.S. ProvisionalApplication No. 60/559,824, filed Apr. 5, 2004; and U.S. ProvisionalApplication No. 60/647,191, filed Jan. 25, 2005. The contents of theseapplications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 7, 2014, is named27154US_CRF_sequencelisting.txt, and is 493,440 bytes in size.

FIELD OF THE INVENTION

The present invention is in the filed of iRNA agents that can inhibitexpression of vascular endothelial growth factor (VEGF). The inventionalso relates to the use of siRNA targeting VEGF sequences to treatconditions or disorders related to unwanted expression of VEGF, e.g.,age-related macular degeneration or diabetic retinopathy.

BACKGROUND

VEGF (also known as vascular permeability factor, VPF) is amultifunctional cytokine that stimulates angiogenesis, epithelial cellproliferation, and endothelial cell survival. VEGF can be produced by awide variety of tissues, and its overexpression or aberrant expressioncan result in a variety disorders, including retinal disorders such asage-related macular degeneration and diabetic retinopathy, cancer,asthma, and other angiogenic disorders.

Macular degeneration is a major cause of blindness in the United Statesand the frequency of this disorder increases with age. Maculardegeneration refers to the group of diseases in which sight-sensingcells in the macular zone of the retina malfunction or loose functionand which can result in debilitating loss of vital central or detailvision. Adult macular degeneration (AMD), which is the most common formof macular degeneration, occurs in two main forms. Ninety percent ofpeople with AMD have the form described as “dry” macular degeneration.An area of the retina is affected, which leads to slow breakdown ofcells in the macula, and a gradual loss of central vision. The otherform of AMD is “wet” macular degeneration. Although only 10% of peoplewith AMD have this type, it accounts for 90% of blindness from thedisease. As dry AMD progresses, new blood vessels may begin to grow andcause “wet” AMD. These new blood vessels often leak blood and fluidunder the macula. This causes rapid damage to the macula that can leadto loss of central vision in a short time. iRNA agents targeting VEGFcan be useful for the treatment of wet and dry macular degeneration.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi has beensuggested as a method of developing a new class of therapeutic agents.However, to date, these have remained mostly as suggestions with nodemonstrate proof that RNAi can be used therapeutically.

The present invention advances the art by providing a detailed gene walkacross the VEGF gene and a detailed structural analysis of modificationsthat can be employed to stabilize the molecule against degradation andincrease cellular uptake and targeting.

SUMMARY OF THE INVENTION

The invention provides compounds, compositions and methods useful formodulating the expression of VEGF. The invention provides compounds,compositions and methods useful for modulating the expression of VEGFactivity by RNA interference (RNAi) using small nucleic acid molecules,such as short interfering RNA (siRNA), double-stranded RNA (dsRNA),microRNA (miRNA) and short hairpin RNA (shRNA) molecules, whichcollectively fall under the general term of iRNA agents. The iRNA agentscan be unmodified or chemically-modified nucleic acid molecules. TheiRNA agents can be chemically synthesized or expressed from a vector orenzymatically synthesized. The invention provides variouschemically-modified synthetic iRNA agents capable of modulating VEGFgene expression or activity in cells and in a mammal by RNAi. The use ofa chemically-modified iRNA agent can improve one or more properties ofan iRNA agent through increased resistance to degradation, increasedspecificity to target moieties, improved cellular uptake, and the like.

In one aspect, the invention provides an iRNA agent that down-regulatesexpression of a VEGF gene. The VEGF gene can include a VEGF encodingsequence and/or VEGF regulatory sequences such as may exist 5′ or 3′ ofa VEGF open reading frame (ORF).

In one embodiment, the invention provides an isolated iRNA agentincluding a sense and antisense sequence, where the sense and antisensesequences can form an RNA duplex. The sense sequence can include anucleotide sequence that is identical or substantially identical to atarget sequence of about 19 to 23 nucleotides of a VEGF sequence. In oneembodiment, the VEGF sequence that is targeted includes the sequence ofany one of SEQ ID NOs:2-401 (see Table 1).

In one embodiment, the sense sequence of the iRNA agent includes asequence identical or substantially identical to any of the VEGF targetsequences, e.g., substantially identical to any of sense sequencesprovided in Table 1, SEQ ID NOs:2-401. In another embodiment, theantisense sequence of the iRNA agent can include a sequencecomplementary to or substantially complementary to, any of the targetsequences, e.g., complementary to any of SEQ ID NOs:2-401. By“substantially identical” is meant that the mismatch between thenucleotide sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.Preferably, no more than 1, 2, 3, 4, or 5 nucleotides differ between thetarget sequence and sense sequence. Furthermore, sequences that are“complementary” to each other (e.g., sense and antisense sequences) canbe fully complementary, or can have no more than 1, 2, 3, 4, or 5nucleotides that lack full complementarity.

In one embodiment, the sense and antisense pairs of sequences of an iRNAagent includes any one of the agents provided in Table 2, or a sequencewhich differs in the sense strand from the recited sequence by no morethan 1, 2, 3, 4, or 5 nucleotides, or in the antisense strand by no morethan 1, 2, 3, 4, or 5 nucleotides, or in both strands by no more than 1,2, 3, 4, or 5 nucleotides.

In one preferred embodiment, the sense sequence of an iRNA agentincludes a sequence that is selected from the group consisting of SEQ IDNO:456, SEQ ID NO:550, SEQ ID NO:608, and SEQ ID NO:634, or a sequencethat differs from the recited sequence by no more than 1, 2, 3, 4, or 5nucleotides.

In another embodiment, the antisense sequence of the iRNA agent includesa sequence fully complementary or substantially complementary to any ofthe VEGF target sequences, e.g., complementary or substantiallycomplementary to any of SEQ ID NOs:2-401.

In another embodiment, the antisense sequence of an iRNA agent includesa sequence selected from the group consisting any of the antisensesequences provided in Table 2, or a sequence which differs from therecited sequence by no more than 1, 2, 3, 4, or 5 nucleotides. In apreferred embodiment, this antisense sequence is fully complementary toa sense sequence or has no more than 1, 2, 3, 4, or 5 nucleotidemismatches with the sense sequence.

In a preferred embodiment, the antisense sequence of an iRNA agentincludes a sequence selected from the group consisting of SEQ ID NO:457,SEQ ID NO:551, SEQ ID NO:609, and SEQ ID NO:635, or a sequence thatdiffers from the recited sequence by no more than 1, 2, 3, 4, or 5nucleotides.

In another embodiment, the iRNA agent is chemically modified. Forexample, the iRNA agent can include a non-nucleotide moiety. A chemicalmodification or other non-nucleotide moiety can stabilize the sense andantisense sequences against nucleolytic degradation. Additionally,conjugates can be used to increase uptake and target uptake of the iRNAagent to particular cell types. Preferred modifications include thosespecifically provided in the Examples, Tables 6-18.

In another embodiment, the iRNA agent includes a 3′-overhang that rangesfrom 1 to about 6 nucleotides. As used herein, a “3′ overhang” refers toat least one unpaired nucleotide extending from the 3′ end of an iRNAsequence. The 3′ overhang can include ribonucleotides ordeoxyribonucleotides or modified ribonucleotides or modifieddeoxyribonucleotides. The 3′ overhang is preferably from 1 to about 5nucleotides in length, more preferably from 1 to about 4 nucleotides inlength and most preferably from about 2 to about 4 nucleotides inlength. The 3′ overhang can occur on the sense or antisense sequence, oron both sequences of an iRNA agent.

In one preferred embodiment, the iRNA agent of the invention includes anantisense sequence having 23 nucleotides complementary to the targetVEGF sequence and a sense sequence having at least 21 nucleotides. Eachsequence can include at least 21 nucleotides that are complementary toeach other, and at least the antisense sequence can have a 3′ overhangof two nucleotides.

In one embodiment, both the sense and antisense sequences of the iRNAagent include a 3′ overhang, the length of which can be the same ordifferent for each sequence. In one embodiment, the 3′ overhang on eachsequence ranges from 1 to about 6 (e.g., from 1 to about 3) nucleotidesin length. In a preferred embodiment, the 3′ overhang is on bothsequences of the iRNA agent and is two nucleotides in length. In anotherpreferred embodiment, the 3′ overhang is on both sequences of the iRNAagent and the 3′ overhangs include two thymidylic acid residues (“TT”).

In one embodiment, an iRNA agent includes an antisense sequence havingabout 19 to 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotideswith complementarity to an RNA sequence encoding a VEGF protein. TheiRNA agent can further include a sense sequence having about 19 to 25(e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides, and theantisense and sense sequences can have distinct nucleotide sequenceswith at least about 19, 20, or 21 complementary nucleotides.

In one embodiment, an iRNA agent of the invention includes an antisenseregion having about 19 to about 25 (e.g., about 19 to about 23)nucleotides with complementarity to an RNA sequence encoding VEGF, and asense region having about 19 to 25 (e.g., about 19 to about 23)nucleotides. The sense and antisense regions can be included in a linearmolecule with at least about 19 complementary nucleotides. The sensesequence can include a nucleotide sequence that is substantiallyidentical to a nucleotide sequence of VEGF.

In one embodiment, the iRNA agent includes an antisense sequence ofabout 21 nucleotides complementary to the VEGF target sequence and asense sequence of about 21 nucleotides complementary to the antisensesequence. The iRNA agent can include a non-nucleotide moiety. In oneembodiment, the sense or antisense sequence of the iRNA agent caninclude a 2′-O-methyl (2′-OMe) pyrimidine nucleotide, 2′-deoxynucleotide (e.g., deoxy-cytodine), 2′-deoxy-2′-fluoro (2′-F) pyrimidinenucleotide, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O—N-methylacetamido (2′-O-NMA), 2′-O-dimethylaminoethlyoxyethyl(2′-DMAEOE), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-AP), 2′-hydroxy nucleotide, or a2′-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extendednucleic acid (ENA), hexose nucleic acid (HNA), cyclohexene nucleic acid(CeNA), ribo-difluorotoluoyl, 5-allyamino-pyrimidines, or5-Me-2′-modified pyrimidines. A 2′ modification is preferably a 2′-OMemodification, and more preferably, a 2′-fluoro modification. In apreferred embodiment, one or more 2′ modified nucleotides are on thesense strand of the iRNA agent.

In one embodiment, an iRNA agent includes a nucleobase modification,such as a cationic modification, such as a 3′-abasic cationicmodification. The cationic modification can be, e.g., an alkylamino-dT(e.g., a C6 amino-dT), an allylamino conjugate, a pyrrolidine conjugate,a pthalamido a hydroxyprolinol conjugate or an aminooxy conjugate, onone or more of the terminal nucleotides of the iRNA agent. Analkylamino-dT conjugate is preferably attached to the 3′ end of thesense or antisense strand of an iRNA agent. A pyrrolidine linker ispreferably attached to the 3′ or 5′ end of the sense strand, or the 3′end of the antisense strand. An allyl amine uridine is preferably on the3′ or 5′ end of the sense strand, and not on the 5′ end of the antisensestrand. An aminooxy conjugate can be attached to a hydroxyl prolinol andat the 3′ or 5′ end of either the sense or antisense strands.

In another embodiment, an iRNA agent that targets VEGF includes aconjugate, e.g., to facilitate entry into a cell or to inhibit exo- orendonucleolytic cleavage. The conjugate can be, for example, alipophile, a terpene, a protein binding agent, a vitamin, acarbohydrate, a retinoid or a peptide. For example, the conjugate can benaproxen, nitroindole (or another conjugate that contributes to stackinginteractions), folate, ibuprofen, retinol or a C5 pyrimidine linker. Inother embodiments, the conjugates are glyceride lipid conjugates (e.g. adialkyl glyceride derivatives), vitamin E conjugates, orthio-cholesterols. Preferably, conjugates are on the 3′ end of theantisense strand, or on the 5′ or 3′ end of the sense strand, andpreferably the conjugates are not on the 3′ end of the antisense strandand on the 3′ end of the sense strand.

In one embodiment, the conjugate is naproxen, and the conjugate ispreferably on the 5′ or 3′ end of the sense or antisense strands. In oneembodiment, the conjugate is cholesterol or thiocholesterol, and theconjugate is preferably on the 5′ or 3′ end of the sense strand andpreferably not present on the antisense strand. In some embodiments, thecholesterol is conjugated to the iRNA agent by a pyrrolidine linker, orserinol linker, or hydroxyprolinol linker. In another embodiment, theconjugate is cholanic acid, and the cholanic acid is attached to the 5′or 3′ end of the sense strand, or the 3′ end of the antisense strand. Inone embodiment, the cholanic acid is attached to the 3′ end of the sensestrand and the 3′ end of the antisense strand. In another embodiment,the conjugate is retinol acid, and the retinol acid is attached to the5′ or 3′ end of the sense strand, or the 3′ end of the antisense strand.In one embodiment, the retinol acid is attached to the 3′ end of thesense strand and the 3′ end of the antisense strand.

In one aspect, an iRNA agent of the invention has RNAi activity thatmodulates expression of RNA encoded by a VEGF gene. VEGF genes can sharesome degree of sequence identity with each other, and thus, iRNA agentscan target a class of VEGF genes, or alternatively, specific VEGF genes,by targeting sequences that are either shared amongst different VEGFtargets or that are unique for a specific VEGF target. Therefore, in oneembodiment, an iRNA agent can target a conserved region of a VEGFnucleotide sequence (e.g., RNA sequence). The conserved region can havesequence identity with several different VEGF-related sequences (e.g.,different VEGF isoforms, splice variants, mutant genes, etc.). Thus, oneiRNA agent can target several different VEGF-related sequences.

In one embodiment, an iRNA agent is chemically modified. In anotherembodiment the iRNA agent includes a duplex molecule wherein one or moresequences of the duplex molecule is chemically modified. Non-limitingexamples of such chemical modifications include phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5′-C-methyl nucleotides, andterminal glyceryl and/or inverted deoxy abasic residue incorporation.These chemical modifications, when used in iRNA agents, can help topreserve RNAi activity of the agents in cells and can increase the serumstability of the iRNA agents.

In one embodiment, an iRNA agent includes one or more chemicalmodifications and the sense and antisense sequences of thedouble-stranded RNA is about 21 nucleotides long.

In a preferred embodiment, the first and preferably the first twointernucleotide linkages at the 5′ end of the antisense and/or sensesequences are modified, preferably by a phosphorothioate. In a preferredembodiment, the first, and preferably the first two, three, or fourinternucleotide linkages at the 3′ end of a sense and/or antisensesequence are modified, preferably by a phosphorothioate. Morepreferably, the 5′ end of both the sense and antisense sequences, andthe 3′ end of both the sense and antisense sequences are modified asdescribed.

In another aspect, an iRNA agent that mediates the down-regulation ofVEGF expression includes one or more chemical modifications thatmodulate the binding affinity between the sense and the antisensesequences of the iRNA construct.

In one embodiment, the invention features an iRNA agent that includesone or more chemical modifications that can modulate the cellular uptakeof the iRNA agent.

In another embodiment, the invention features an iRNA agent thatincludes one or more chemical modifications that improve thepharmacokinetics of the iRNA agent. Such chemical modifications includebut are not limited to conjugates, such as ligands for cellularreceptors, e.g., peptides derived from naturally occurring proteinligands; protein localization sequences; antibodies; nucleic acidaptamers; vitamins and other co-factors, such as folate, retinoids andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG, e.g.PEG 5 and PEG20); phospholipids; polyamines, such as spermine orspermidine; and others.

In one embodiment, the iRNA agent includes a duplex molecule selectedfrom the group consisting of AL-DP-4003, AL-DP-4116, AL-DP-4015,AL-DP-4120, AL-DP-4002, AL-DP-4115, AL-DP-4014, AL-DP-4119, AL-DP-4094,AL-DP-4118, AL-DP-4107, AL-DP-4122, AL-DP-4004, AL-DP-4117, AL-DP-4016,AL-DP-4121, AL-DP-4127, AL-DP-4128, AL-DP-4129, and AL-DP-4055 (seeTables 2 and 3).

In one preferred embodiment, the iRNA agent includes a duplex describedas AL-DP-4094, which includes the antisense sequence5′AAGCUCAUCUCUCCUAUGUGCUG 3′ (SEQ ID NO:609) and the sense sequence 5′GCACAUAGGAGAGAUGAGCUU 3′ (SEQ ID NO:608).

In another preferred embodiment, the iRNA agent includes a duplexdescribed as AL-DP-4004, which includes the antisense sequence5′CUUUCUUUGGUCUGCAUUCACAU 3′ (SEQ ID NO:635) and the sense sequence 5′GUGAAUGCAGACCAAAGAAAG 3′ (SEQ ID NO:634).

In another preferred embodiment, the iRNA agent includes a duplexdescribed as AL-DP-4015, which includes the antisense sequence 5′GUACUCCUGGAAGAUGUCCTT 3′ (SEQ ID NO:551) and the sense sequence 5′GGACAUCUUCCAGGAGUACTT 3′ (SEQ ID NO:550).

In another preferred embodiment, the iRNA agent includes a duplexdescribed as AL-DP-4055, which includes the antisense sequence 5′UGCAGCCUGGGACCACUUGTT 3′ (SEQ ID NO:457) and the sense sequence 5′CAAGUGGUCCCAGGCUGCATT 3′ (SEQ ID NO:456).

In one embodiment, the antisense sequence of an iRNA agent describedherein does not hybridize to an off-target sequence. For example, theantisense sequence can have less than 5, 4, 3, 2, or 1 nucleotidescomplementary to an off-target sequence. By “off-target” is meant asequence other than a VEGF nucleotide sequence.

In another embodiment, the sense strand is modified to inhibitoff-target silencing. The sense strand can include a cholesterol moeity,such as cholesterol attached to the sense strand by a pyrrolidinelinker.

In another embodiment, the antisense sequence of an iRNA agent describedherein can hybridize to a VEGF sequence in a human and a VEGF sequencein a non-human mammal, e.g., a mouse, rat, or monkey.

In another aspect, the invention provides a method of delivering an iRNAagent, e.g., an iRNA agent described herein, to the eye of a subject,e.g., a mammalian subject, such as a mouse, a rat, a monkey or a human.

In one embodiment, the iRNA agent can be delivered to a cell or cells ina choroid region of the eye. In one preferred embodiment, the iRNA agentdown-regulates expression of the VEGF gene at a target site within theeye. An iRNA agent delivered to the eye, e.g., choroid cells of the eye,can be an unmodified iRNA agent.

In one embodiment, the iRNA agent can be stabilized withphosphorothioate linkages. In another embodiment, the 3′ end of thesense or antisense sequences, or both, of the iRNA agent can be modifiedwith a cationic group, such as a 3′-abasic cationic modification. Thecationic modification can be, e.g., an alkylamino-dT (e.g., a C6amino-dT), an allylamine, a pyrrolidine, a pthalamido, ahydroxyprolinol, a polyamine, a cationic peptide, or a cationic aminoacid on one or more of the terminal nucleotides of the iRNA agent. Themodification can be an external or terminal cationic residue. Inpreferred embodiments, a pyrrolidine cap is attached to the 3′ or 5′ endof the sense strand, or the 3′ end of the antisense strand.

In one embodiment, the sense or antisense sequence, or both, of the iRNAagent can be modified with a sugar, e.g., a glycoconjugate oralkylglycoside component, e.g., glucose, mannose, 2-deoxy-glucose, or ananalog thereof. In another embodiment, the iRNA agent can be conjugatedto an enzyme substrate, e.g., a substrate for which the relative enzymeis present in a higher amount, as compared to the enzyme level in othertissues of the body, e.g., in tissues other than the eye.

In one embodiment, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore of the iRNA agent administered to the subject reaches the eye. In apreferred embodiment, between about 30-90%, 40-80% or 50-70% of the iRNAagent administered to the subject reaches the eye.

In another aspect, the invention features a composition, e.g., apharmaceutical composition that includes an iRNA agent of the presentinvention in a pharmaceutically acceptable carrier or diluent. The iRNAagent can be any agent described herein. In one embodiment, the iRNAagent is chemically modified, such as with any chemical modificationdescribed herein. Preferred modified iRNA agents includes those providedin Tables 2-18.

In another aspect, the invention features a method for treating orpreventing a disease or condition in a subject. The method can includeadministering to the subject a composition of the invention underconditions suitable for the treatment or prevention of the disease orcondition in the subject, alone or in conjunction with one or more othertherapeutic compounds.

In one embodiment, the iRNA agent is administered at or near the site ofunwanted VEGF expression, e.g., by a catheter or other placement device(e.g., a retinal pellet or an implant including a porous, non-porous, orgelatinous material). In one embodiment the iRNA agent is administeredvia an intraocular implant, which can be inserted, for example, into ananterior or posterior chamber of the eye; or into the sclera,transchoroidal space, or an avascularized region exterior to thevitreous. In another embodiment, the implant is positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g., to theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion is preferably in proximity to the macula.

In another embodiment, an iRNA agent is administered to the eye byinjection, e.g., by intraocular, retinal, or subretinal injection.

In another embodiment, an iRNA agent is administered topically to theeye, such as by a patch or liquid eye drops, or by iontophoresis.Ointments or droppable liquids can be delivered by ocular deliverysystems known in the art such as applicators or eye droppers.

In one embodiment, an iRNA is delivered at or near a site ofneovascularization.

In one embodiment, an iRNA agent is administered repeatedly.Administration of an iRNA agent can be carried out over a range of timeperiods. It can be administered hourly, daily, once every few days,weekly, or monthly. The timing of administration can vary from patientto patient, depending upon such factors as the severity of a patient'ssymptoms. For example, an effective dose of an iRNA agent can beadministered to a patient once a month for an indefinite period of time,or until the patient no longer requires therapy. In addition, sustainedrelease compositions containing an iRNA agent can be used to maintain arelatively constant dosage in the area of the target VEGF nucleotidesequences.

In another embodiment, an iRNA agent is delivered to the eye at a dosageon the order of about 0.00001 mg to about 3 mg per eye, or preferrablyabout 0.0001-0.001 mg per eye, about 0.03-3.0 mg per eye, about 0.1-3.0mg per eye or about 0.3-3.0 mg per eye.

In another embodiment, an iRNA agent is administered prophylacticallysuch as to prevent or slow the onset of a disorder or condition thataffects the eye. For example, an iRNA can be administered to a patientwho is susceptible to or otherwise at risk for a neovascular disorder.

In one embodiment one eye of a human is treated with an iRNA agentdescribed herein, and in another embodiment, both eyes of a human aretreated.

In another aspect, a method of inhibiting VEGF expression is provided.One such method includes administering an effective amount of an iRNAagent of the present invention.

In another aspect, a method of treating adult onset macular degenerationis provided. The method includes administering a therapeuticallyeffective amount of an iRNA agent of the present invention.

In one embodiment, a human has been diagnosed with dry adult maculardegeneration (AMD), and in another embodiment the human has beendiagnosed with wet AMD.

In one embodiment, a human treated with an iRNA agent described hereinis over the age of 50, e.g., between the ages of 75 and 80, and thehuman has been diagnosed with adult onset macular degeneration. Inanother embodiment, a human treated with an iRNA agent described hereinis between the ages of 30-50, and the human has been diagnosed with lateonset macular degeneration. In another embodiment, a human treated withan iRNA agent described herein is between the ages of 5-20, and thehuman has been diagnosed with middle onset macular degeneration. Inanother embodiment, a human treated with an iRNA agent described hereinis 7 years old or younger, and the human has been diagnosed with earlyonset macular degeneration.

In one aspect, methods of treating any disease or disorder characterizedby unwanted VEGF expression are provided. Particularly preferredembodiments include the treatment of disorders of the eye or retina,which are characterized by unwanted VEGF expression. The disease ordisorder can be a diabetic retinopathy, neovascular glaucoma, a tumor ormetastic cancer (e.g., colon or breast cancer), a pulmonary disease(e.g., asthma or bronchitis), rheumatoid arthritis, or psoriases. Otherangiogenic disorders can be treated by the methods featured in theinvention.

In another aspect, the invention features a kit containing an iRNA agentof the invention. The iRNA agent of the kit can be chemically modifiedand can be useful for modulating the expression of a VEGF target gene ina cell, tissue or organism. In one embodiment, the kit contains morethan one iRNA agent of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, useful methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the invention will be apparent from the accompanyingdrawings and description, and from the claims. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference. In case of conflict, the present specification, includingdefinitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of the mRNA of the 121 amino acid formof vascular endothelial growth factor, VEGF121. The first nucleotide ofthe initiator codon is nucleotide 1. The signal peptide is fromnucleotide 1 through 78.

FIG. 2 is a graphical representation of a comparative analysis of theactivities of single- and double-overhang siRNAs in in vitro assays inHeLa cells. Solid lines with filled symbols represent thesingle-overhang siRNA, solid lines with open symbols represent thedouble-overhang siRNAs; dashed lines represent the control siRNAs. Thecontrol siRNA hVEGF is described in Reich et al. (Mol. Vis. 9:210,2003); the control siRNA hrmVEGF is described in Filleur et al. (CancerRes. 63:3919, 2003). “L2000” refers to Lipofectamine 2000 reagent. hVEGFexpression (y-axis) refers to endogenous VEGF expression.

FIG. 3 is a graphical representation of a comparative analysis of theactivities of single- and double-overhang siRNAs in ARPE-19 cells. Solidlines with filled symbols represent the single-overhang siRNA; solidlines with open symbols represent the double-overhang siRNAs; dashedlines represent the control siRNAs. The control siRNA hVEGF is describedin Reich et al. (Mol. Vis. 9:210, 2003); the control siRNA hrmVEGF isdescribed in Filleur et al. (supra). “L2000” refers to Lipofectamine2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGFexpression.

FIG. 4 is a graphical representation of a comparative analysis of thesiRNAs activities in HeLa cells of single-overhang siRNAs with theiranalogous blunt siRNAs in which the number of base-paired nucleotides is21. The control siRNA hVEGF is described in Reich et al. (Mol. Vis.9:210, 2003); the control siRNA hrmVEGF is described in Filleur et al.(supra). “L2000” refers to Lipofectamine 2000 reagent. hVEGF expression(y-axis) refers to endogenous VEGF expression.

FIG. 5 is a graphical representation of a comparative analysis of thesiRNAs activities in HeLa cells of double-overhang siRNAs with theiranalogous blunt siRNAs in which the number of base-paired nucleotides is19. The control siRNA hVEGF is described in Reich et al. (supra); thecontrol siRNA hrmVEGF is described in Filleur et al. (supra). “L2000”refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refersto endogenous VEGF expression.

FIG. 6A is a graphical representation of the activities ofsingle-overhang and double overhang siRNAs targeting ORF 319 (SEQ IDNO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ IDNO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under normaloxygen (normoxia, 20% oxygen).

FIG. 6B is a graphical representation of the activities ofsingle-overhang and double overhang siRNAs targeting ORF 319 (SEQ IDNO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ IDNO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under hypoxicconditions (1% oxygen).

FIG. 6C is a graphical representation of the activities ofsingle-overhang and double overhang siRNAs targeting ORF 319 (SEQ IDNO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ IDNO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under hypoxicconditions (130 μM defoxamine).

FIG. 7 is a graphical representation of the comparative activities ofdouble-overhang (AL-DP-4014) unmodified siRNA andphosphorothioate-modified (AL-DP-4127, AL-DP-4128, AL-DP-4129) siRNAstargeting ORF 319 (SEQ ID NO:320) in HeLa cells. The control siRNA hVEGFis described in Reich et al. (supra); the control siRNA hrmVEGF isdescribed in Filleur et al. (supra). “L2000” refers to Lipofectamine2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGFexpression.

FIG. 8A is a graphical representation of the activities of siRNAstargeting ORF 319 (SEQ ID NO:320) (AL-DP-4014 and AL-DP-4127) and amutated version AL-DP-4140 (Table 5) in cells under normal oxygenconditions (normoxia, 20% oxygen). The control siRNA Cand5 is identicalto the hVEGF control of FIG. 7 and is described in Reich et al. (supra).“L2000” refers to Lipofectamine 2000 reagent. VEGF expression (y-axis)refers to endogenous VEGF expression.

FIG. 8B is a graphical representation of the activities of siRNAstargeting ORF 319 (SEQ ID NO:320) (AL-DP-4014 and AL-DP-4127) and amutated version AL-DP-4140 (Table 5) in cells under normal or hypoxicconditions (hypoxia, 1% Oxygen). The control siRNAs are as described forFIG. 8A.

FIG. 9A is a graphical representation of the activities of siRNAs havingthe sequence of AL-DP-4094 but differing in the inclusion of nucleotidemodifications (see Table 4). FIG. 9A sense strands disclosed as SEQ IDNO: 652 and antisense strands disclosed as SEQ ID NOS 653-658,respectively in order of appearance.

FIG. 9B is a graphical representation of the activities of siRNAs havingthe sequence of AL-DP-4094 but differing in the inclusion of nucleotidemodifications (see Table 4). FIG. 9B sense strands disclosed as SEQ IDNO: 659 and antisense strands disclosed as SEQ ID NOS 653-658,respectively in order of appearance.

FIG. 9C is a graphical representation of the activities of siRNAs havingthe sequence of AL-DP-4094 but differing in the inclusion of nucleotidemodifications (see Table 4). FIG. 9C sense strands disclosed as SEQ IDNO: 660 and antisense strands disclosed as SEQ ID NOS 653-658,respectively in order of appearance.

FIG. 9D is a graphical representation of the activities of siRNAs havingthe sequence of AL-DP-4094 but differing in the inclusion of nucleotidemodifications (see Table 4). FIG. 9D sense strands disclosed as SEQ IDNO: 661 and antisense strands disclosed as SEQ ID NOS 653-658,respectively in order of appearance.

FIG. 9E is a graphical representation of the activities of siRNAs havingthe sequence of AL-DP-4094 but differing in the inclusion of nucleotidemodifications (see Table 4). FIG. 9E sense strands disclosed as SEQ IDNOS 662-665, 652, 652 and 652 and antisense strands disclosed as SEQ IDNO: 653, 653, 653, 653 and 666-668, respectively in order of appearance.The control siRNA “Acuity” is identical to the Cand5 control of FIG. 8Aand the hVEGF control of FIG. 7. The “Filleur” control siRNA is theequivalent of the hrmVEGF control siRNA of FIG. 7.

FIG. 10 is a graphical representation of siRNA silencing activity invitro in HeLa cells.

FIG. 11 is an RP-HPLC scan of AL-DP-4094 siRNA following incubation inhuman serum.

FIG. 12 is a summary of AL-DP-4094 fragment mapping as determined byLC/MS. The analysis was performed following incubation of the siRNA inhuman serum (SEQ ID NOS 608-609, 1080-1082, 1082-1083, 608, 611, 611 and1084, respectively, in order of appearance)

FIG. 13 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 14 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 15 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 16 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 17 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 18 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 19 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 20 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 21 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 22 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 23 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 24 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 25 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 26 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 27 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 28 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 29 is a graph of silencing activity of 2′-O-methyl and/or 2′-fluoromodified siRNAs in vitro in HeLa cells (Table 6).

FIG. 30A is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30B is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30C is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30D is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30E is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30F is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30G is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30H is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 30I is a graph of silencing activity of alternating 2′-O-methyl and2′-fluoro modified siRNAs in vitro in HeLa cells (Table 7).

FIG. 31 is a graph of silencing activity of cholesterol and colonicconjugated siRNAs in vitro in HeLa cells (Table 8).

FIG. 32 is a graph of silencing activity of cholesterol and colonicconjugated siRNAs in vitro in HeLa cells (Table 8).

FIG. 33 is a graph of silencing activity of cholesterol and colonicconjugated siRNAs in vitro in HeLa cells (Table 8).

FIG. 34 is a graph of silencing activity of naproxen conjugated siRNAsin vitro in HeLa cells (Table 9).

FIG. 35 is a graph of silencing activity of biotin conjugated siRNAs invitro in HeLa cells (Table 10).

FIG. 36 is a graph of silencing activity of 5′-retinal conjugated siRNAsin vitro in HeLa cells (Table 11).

FIG. 37 is a graph of silencing activity of ribo-difluorotoluoylmodified siRNAs in vitro in HeLa cells (Table 13).

FIG. 38 is a graph of silencing activity of 2′-arafluoro-2′deoxy-nucleoside modified siRNAs in vitro in HeLa cells (Table 14).

FIG. 39 5′-O-DMTr-2′-deoxy-2′-fluoro A, C, G and U CPG supports foroligonucleotide synthesis. These supports were used for syntheses ofselected sequences listed Tables 6 and 7.

FIG. 40 Cholesterol and 5β-cholanic (or cholanic) acid conjugatebuilding blocks for conjugation to oligonucleotides. These buildingblocks were used for syntheses of selected sequences listed in Table 8.

FIG. 41 ^(5Me)C and ^(5Me)U RNA building blocks for oligonucleotidesynthesis. These building blocks were used for syntheses of selectedsequences listed in Table 8.

FIG. 42. Naproxen—trans-4-hydroxy-L-prolinol and naproxen-serinolbuilding blocks for conjugation to oligonucleotides. These buildingblocks were used for syntheses of selected sequences listed in Table 9.

FIG. 43 Biotin—trans-4-hydroxy-L-prolinol and biotin-serinol buildingblocks for conjugation to oligonucleotides. These building blocks wereused for syntheses of selected sequences listed in Table 10.

FIG. 44 Building blocks for post-synthetic conjugation—Oxime approach.These building blocks were/are used for syntheses of selected sequenceslisted in Table 11.

FIG. 45 Building blocks for post-synthetic conjugation—Active esterapproach. These building blocks were used for syntheses of selectedsequences listed in Table 12.

FIG. 46 DFT amidite and CPG for oligonucleotide synthesis. Thesebuilding blocks were used for syntheses of selected sequences listed inTable 13.

FIG. 47 2′-Deoxy-2′-araf amidite for oligonucleotide synthesis. Thesebuilding blocks were used for syntheses of selected sequences listed inTable 14.

FIG. 48 P-methylphosphonamidite of ribo ^(5Me)U and ribo C(N^(Ac)).These building blocks were used for syntheses of selected sequenceslisted in Table 15.

FIG. 49 C5-aminoallyl U amidite. These building blocks were used forsyntheses of selected sequences listed in Table 16.

FIG. 50 Thiocholesterol conjugate building blocks.

FIG. 51 is a graphical representation of VEGF protein levels in HeLacells following administration of the indicated double-stranded RNAs.

FIG. 52 is a graphical representation of VEGF protein levels in ARPE-19cells following administration of the indicated double-stranded RNAs.

FIG. 53 is a graphical representation of VEGF protein levels in ratRPE-J cells following administration of the indicated double-strandedRNAs.

FIG. 54 is a graphical representation of VEGF mRNA levels in HeLa cellsfollowing administration of the indicated double-stranded RNAs.Expression levels were measured according to the ratio of VEGF mRNA to aGAPDH control mRNA.

FIG. 55 is a bar graph illustrating the effect of varying concentrationsof the indicated double-stranded RNAs at 5 and 10 dayspost-transfection.

FIG. 56 is a bar graph illustrating the area of pathologicneovascularization in mouse retinas following administration of theindicated agents. “Buffer” is phosphate buffered saline (PBS); “control”is a double-stranded RNA targeting luciferase (AL-DP-3015); “VEGF” is adouble-stranded RNA targeting VEGF (AL-DP-4409).

FIG. 57 is a bar graph illustrating the effect of the indicated agentson normal vascularization in mouse retinas. “Buffer” is phosphatebuffered saline (PBS); “control” is a double-stranded RNA targetingluciferase (AL-DP-3015); “VEGF” is a double-stranded RNA targeting VEGF(AL-DP-4409).

FIG. 58 is a bar graph illustrating the area of pathologicneovascularization in mouse retinas following administration of theindicated agents. “PBS” is phosphate buffered saline; “siMM” isAL-DP-4409 mM, a double-stranded RNA including a mismatch sequence ascompared to the double-stranded RNA (siRNA), AL-DP-4409, that targetsVEGF; “siVEGF” is AL-DP-4409.

FIG. 59 is a bar graph illustrating the effect of the indicated agentson normal vascularization in mouse retinas. “PBS” is phosphate bufferedsaline; “siMM” is AL-DP-4409 mM, a double-stranded RNA including amismatch sequence as compared to the double-stranded RNA (siRNA),AL-DP-4409, that targets VEGF; “siVEGF” is AL-DP-4409.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides the sequences in the VEGF gene that are targeted by theagents of the present invention. These sequence can also be the sensestrand of some of the iRNA agents of the present invention.

Table 2 provides 123 iRNA duplexes that target the VEGF gene, the targetsequence in the VEGF gene and activity data that is described in theExamples.

Table 3 provides iRNA duplexes that are modified to containphosphorothioate stabilizations and activity data that is described inthe Examples.

Table 4 provides iRNA duplexes based on the AL-DP-4094 duplex that aremodified for stabilization and activity data that is described in theExamples.

Table 5 provides iRNA duplexes activity data in HeLa cells for severaliRNA agents of the present invention.

Table 6 provides iRNA agents with activity data in HeLa cells for agentscontaining one or more phosphorothioate, 2′-O-methyl and 2′-fluoromodifications.

Table 7 provides iRNA agents with activity data in HeLa cells for agentscontaining alternating 2′-O-methyl and 2′-fluoro modifications.

Table 8 A and B provides iRNA agents with activity data in HeLa cellsfor agents containing cholesterol or cholanic acid conjugates.

Table 9 provides iRNA agents with activity data in HeLa cells for agentscontaining naproxen conjugates.

Table 10 provides iRNA agents with activity data in HeLa cells foragents containing biotin conjugates.

Table 11 provides iRNA agents containing aldehydes, retinal and otherretinoid conjugates.

Table 12 provides iRNA agents containing polyethylene glycol conjugates.

Table 13 provides iRNA agents with activity data in HeLa cells foragents containing ribo-difluorotoluoyl modifications.

Table 14 provides iRNA agents with activity data in HeLa cells foragents containing 2′-arafluoro-2′-deoxy-nucleoside modifications.

Table 15 provides iRNA agents containing methylphosphonatemodifications.

Table 16 provides iRNA agents containing C-5 allyamino modifications.

Table 17 provides iRNA agents containing a variety and combinations ofthe modifications as noted in the Table.

Table 18 provides physical characterization of iRNA agents containing avariety and combinations of the modifications as noted in the Table.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments recruits cellular factorsthat mediate RNAi. Described herein are methods for preparing andadministering these 21-23 nt fragments, and other iRNAs agents, andtheir use for specifically inactivating gene function. The use of iRNAagents (or recombinantly produced or chemically synthesizedoligonucleotides of the same or similar nature) enables the targeting ofspecific mRNAs for silencing in mammalian cells. In addition, longerdsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse, which is frequently deleterious, siRNAs do not trigger theinterferon response, at least not to an extent that is deleterious tothe cell and host. In particular, the length of the sense and antisensesequences in an iRNA agent can be less than 31, 30, 28, 25, or 23 nt,e.g., sufficiently short to avoid inducing a deleterious interferonresponse. Thus, the administration of a composition of iRNA agents(e.g., formulated as described herein) to a mammalian cell can be usedto silence expression of a target gene while circumventing theinterferon response. Further, use of a discrete species of iRNA agentcan be used to selectively target one allele of a target gene, e.g., ina subject heterozygous for the allele.

The target-complementary sequence (the antisense sequence) of an iRNAagent, such as an iRNA duplex, can have a 5′ phosphate and ATP may beutilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen etal., Cell 107:309, 2001); however, iRNA agents lacking a 5′-phosphatehave been shown to be active when introduced exogenously, suggestingthat 5′-phosphorylation of siRNA constructs may occur in vivo.

Vascular Endothelial Growth Factor (VEGF)

VEGF, also known as vascular permeability factor, is an angiogenicgrowth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists inat least three different isoforms. VEGF isoforms are expressed inendothelial cells. The VEGF gene contains 8 exons that express a189-amino acid protein isoform. A 165-amino acid isoform lacks theresidues encoded by exon 6, whereas a 121-amino acid isoform lacks theresidues encoded by exons 6 and 7. VEGF145 is an isoform predicted tocontain 145 amino acids and to lack exon 7.

VEGF can act on endothelial cells by binding to an endothelial tyrosinekinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2is expressed in endothelial cells and is involved in endothelial celldifferentiation and vasculogenesis. A third receptor, VEGFR-3 has beenimplicated in lymphogenesis.

The various isoforms have different biologic activities and clinicalimplications. For example, VEGF145 induces angiogenesis and like VEGF189(but unlike VEGF165) VEGF145 binds efficiently to the extracellularmatrix by a mechanism that is not dependent on extracellularmatrix-associated heparin sulfates. The mRNA corresponding to the codingsequence of human VEGF121 (Genbank Accession Number AF214570, SEQ IDNO:1) is shown in FIG. 1. VEGF displays activity as an endothelial cellmitogen and chemoattractant in vitro and induces vascular permeabilityand angiogenesis in vivo. VEGF is secreted by a wide variety of cancercell types and promotes the growth of tumors by inducing the developmentof tumor-associated vasculature. Inhibition of VEGF function has beenshown to limit both the growth of primary experimental tumors as well asthe incidence of metastases in immunocompromised mice. VEGF is alsoexpressed at abnormally high levels in inflammatory diseases such asrheumatoid arthritis and psoriasis, and is involved in the inflammation,airway and vascular remodeling that occurs during asthmatic episodes.Elevated VEGF expression is also correlated with several forms of ocularneovascularization that often lead to severe vision loss, includingdiabetic retinopathy, retinopathy of prematurity, and maculardegeneration.

iRNA Agents

An “RNA agent,” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate. Preferred examples include those which havegreater resistance to nuclease degradation than do unmodified RNAs.Preferred examples include those which have a 2′ sugar modification, amodification in a single strand overhang, preferably a 3′ single strandoverhang, or, particularly if single stranded, a 5′ modification whichincludes one or more phosphate groups or one or more analogs of aphosphate group.

An “iRNA agent,” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group.

The iRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate down regulationof the target gene. (For ease of exposition the term nucleotide orribonucleotide is sometimes used herein in reference to one or moremonomeric subunits of an RNA agent. It will be understood herein thatthe usage of the term “ribonucleotide” or “nucleotide,” herein can, inthe case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions.) Thus, the iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of the targetRNA, e.g., mRNA.

Complementarity, or degree of homology with the target strand, is mostcritical in the antisense strand. While perfect complementarity,particularly in the antisense strand, is often desired some embodimentscan include, particularly in the antisense strand, one or more butpreferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to thetarget RNA). The mismatches, particularly in the antisense strand, aremost tolerated in the terminal regions and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the overall doublestrand character of the molecule.

Single stranded regions of an iRNA agent will often be modified orinclude nucleoside surrogates, e.g., the unpaired region or regions of ahairpin structure, e.g., a region which links two complementary regions,can have modifications or nucleoside surrogates. Modification tostabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., againstexonucleases, or to favor the antisense sRNA agent to enter into RISCare also favored. Modifications can include C3 (or C6, C7, C12) aminolinkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3,C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), specialbiotin or fluorescein reagents that come as phosphoramidites and thathave another DMT-protected hydroxyl group, allowing multiple couplingsduring RNA synthesis.

iRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.,Nature 409:363-366, 2001)) and enter a RISC (RNAi-induced silencingcomplex); and molecules that are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent” as used herein, refers to aniRNA agent, e.g., a double stranded RNA agent or single strand agent,that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The sRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of a sRNA agent can be equal to or less than 30, 25, 24, 23,22, 21, or 20 nucleotides in length. The strand is preferably at least19 nucleotides in length. For example, each strand can be between 21 and25 nucleotides in length. Preferred sRNA agents have a duplex region of17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or moreoverhangs, preferably one or two 3′ overhangs, of 2-3 nucleotides.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enterthe RISC and participate in RISC mediated cleavage of a target mRNA. Asingle strand iRNA agent is at least 14, and more preferably at least15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferablyless than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agentwhich includes more than one, and preferably two, strands in whichinterchain hybridization can form a region of duplex structure.

Other suitable modifications to a sugar, base, or backbone of an iRNAagent are described in co-owned PCT Application No. PCT/US2004/01193,filed Jan. 16, 2004. An iRNA agent can include a non-naturally occurringbase, such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Many of these types of modifications are provided in the Examples andare described in Tables 3-18.

Design of iRNA.

The present invention is based on a gene walk of the VEGF gene toidentify active iRNA agents that can be used to reduce the level of VEGFmRNA in a cell. Not all potential iRNA agent sequences in the VEGF geneare active, many of which also having significant off-target effects.The present invention advances the art by selecting those sequenceswhich are active and do not have significant off-target effects.Further, the sequence chosen for the iRNA agents of the presentinvention are conserved amongst multiple species allowing one to use asingle agent for animal and toxicological studies as well as using itfor therapeutic purposes in humans.

Based on these results, the invention specifically provides an iRNAagent that can be used in treating VEGF mediated disorders, particularlyin the eye such as AMD, in isolated form and as a pharmaceuticalcomposition described below. Such agents will include a sense strandhaving at least 15 or more contiguous nucleotides that are complementaryto the VEGF gene and an antisense strand having at least 15 or morecontiguous nucleotides that are complementary to the sense strandsequence. Particularly useful are iRNA agents that have a sense strandthat comprises, consist essentially of or consists of a nucleotidesequence provided in Table 1, such as those agents proved in Table 2, orany of the modifications provided in Tables 3-18.

Candidate iRNA agents can be designed by performing, as done herein, agene walk analysis of the VEGF gene that will serve as the iRNA target.Overlapping, adjacent, or closely spaced candidate agents correspondingto all or some of the transcribed region can be generated and tested.Each of the iRNA agents can be tested and evaluated for the ability todown regulate the target gene expression (see below, “Evaluation ofCandidate iRNA agents”).

Preferably, the iRNA agents of the present invention are based on andcomprise at least 15 or more contiguous nucleotides from one of the iRNAagents shown to be active in Table 2, or the modified sequences providedin Tables 3-18. In such agents, the agent can comprise, consist of orconsist essentially of the entire sequence provided in the Table or cancomprise 15 or more contiguous residues along with additionalnucleotides from contiguous regions of the target gene.

An iRNA agent can be rationally designed based on sequence informationand desired characteristics and the information of the target sequenceprovided in Table 1. For example, an iRNA agent can be designedaccording to the relative melting temperature of the candidate duplex.Generally, the duplex should have a lower melting temperature at the 5′end of the antisense strand than at the 3′ end of the antisense strand.

Accordingly, the present invention provides iRNA agents comprising asense strand and antisense strand each comprising a sequence of at least15, 16, 17, 18, 19, 20, 21 or 23 nucleotides which is essentiallyidentical to one of the agents provided in Table 1 or 2.

The antisense strand of an iRNA agent should be equal to or at least,15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should beequal to or less than 50, 40, or 30, nucleotides in length. Preferredranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the agents in Table 2 (or are complementary tothe target sequence provided in Table 1) but are not longer than 25nucleotides in length.

The sense strand of an iRNA agent should be equal to or at least 15, 1617, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equalto or less than 50, 40, or 30 nucleotides in length. Preferred rangesare 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.Exemplified iRNA agents include those that comprise 15 or morenucleotides from one of the agents in Table 2 (or the target sequence inTable 2) but are not longer than 25 nucleotides in length.

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50nucleotide pairs in length. It should be equal to or less than 50, 40,or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25,19 to 23, and 19 to 21 nucleotides pairs in length.

The agents provided in Table 2 are 23 nucleotides in length for eachstrand. The iRNA agents contain a 21 nucleotide double stranded regionwith a 2 nucleotide overhang on each of the 3′ ends of the agent. Theseagents can be modified as described herein to obtain equivalent agentscomprising at least a portion of these sequences (15 or more contiguousnucleotides) and or modifications to the oligonucleotide bases andlinkages. Particularly preferred are the modification and agentsprovided in Tables 3-18.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the VEGF gene and are of sufficient lengthin terms of nucleotides that the iRNA agent, or a fragment thereof, canmediate down regulation of the VEGF gene. The antisense strands of theiRNA agents of the present invention are preferably fully complementaryto the mRNA sequences of VEGF gene. However, it is not necessary thatthere be perfect complementarity between the iRNA agent and the target,but the correspondence must be sufficient to enable the iRNA agent, or acleavage product thereof, to direct sequence specific silencing, e.g.,by RNAi cleavage of a VEGF mRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of the VEGF gene,such as those agent provided in Table 2, except that not more than 1, 2or 3 nucleotides per strand, respectively, have been substituted byother nucleotides (e.g. adenosine replaced by uracil), while essentiallyretaining the ability to inhibit VEGF expression. These agents willtherefore possess at least 15 or more nucleotides identical to the VEGFgene but 1, 2 or 3 base mismatches with respect to either the VEGF mRNAsequence or between the sense and antisense strand are introduced.Mismatches to the target VEGF mRNA sequence, particularly in theantisense strand, are most tolerated in the terminal regions and ifpresent are preferably in a terminal region or regions, e.g., within 6,5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, most preferablywithin 6, 5, 4, or 3 nucleotides of the 5′-terminus of the sense strandor the 3′-terminus of the antisense strand. The sense strand need onlybe sufficiently complementary with the antisense strand to maintain theoverall double stranded character of the molecule.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule, such as those exemplified in Table 2 (as wellas Tables 3-18). Thus, an iRNA agent contains sense and antisensestrands, preferably paired to contain an overhang, e.g., one or two 5′or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Mostembodiments will have a 3′ overhang. Preferred siRNA agents will havesingle-stranded overhangs, preferably 3′ overhangs, of 1 to 4, orpreferably 2 or 3 nucleotides, in length, on one or both ends of theiRNA agent. The overhangs can be the result of one strand being longerthan the other, or the result of two strands of the same length beingstaggered. 5′-ends are preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. Embodiments in which the twostrands of the siRNA agent are linked, e.g., covalently linked are alsoincluded. Hairpin, or other single strand structures which provide therequired double stranded region, and preferably a 3′ overhang are alsowithin the invention.

Synthesis of iRNA Agents.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) can be synthesized usingprotocols known in the art, for example as described in Caruthers etal., Methods in Enzymology 211:3, 1992; Thompson et al., InternationalPCT Publication No. WO 99/54459; Wincott et al., Nucleic Acids Res.23:2677, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997; Brennan etal., Biotechnol. Bioeng. 61:33, 1998; and Brennan, U.S. Pat. No.6,001,311. All of these references are incorporated herein by reference.The synthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end.

The method of synthesis used for RNA including certain iRNA agents ofthe invention follows the procedure as described in Usman et al., J.Chem. Soc. 109:7845, 1987; Scaringe et al., Nucleic Acids Res. 18:5433,1990; Wincott et al., Nucleic Acids Res. 23:2677, 1995; and Wincott etal., Methods Mol. Bio. 74:59, 1997; and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. Detailed descriptions of a varietyof synthetic methods to produce modified iRNA agents are provided in theExamples.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., Science 256:9923, 1992; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,Nucleic Acids Res. 19:4247, 1991; Bellon et al., Nucleosides &Nucleotides 16:951, 1997; Bellon et al., Bioconjugate 8:204, 1997), orby hybridization following synthesis and/or deprotection.

An iRNA agent can also be assembled from two distinct nucleic acidsequences or fragments wherein one fragment includes the sense regionand the second fragment includes the antisense region of the iRNA agent.iRNA agents can be modified extensively to enhance stability bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-fluoro, difluorortoluoyl, 5-allyamino-pyrimidines,2′-O-methyl, 2′-H (for a review see Usman and Cedergren, Trends inBiochem. Sci. 17:34, 1992). iRNA constructs can be purified by gelelectrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, iRNA agents can be expressed fromtranscription units inserted into DNA or RNA vectors. The recombinantvectors can be DNA plasmids or viral vectors. iRNA agent-expressingviral vectors can be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the iRNA agents can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of iRNA agents.

Evaluating iRNA Agents.

Any of the iRNA agents described herein can be evaluated and modified asfollows.

An iRNA agent may be susceptible to cleavage by an endonuclease orexonuclease, such as when the iRNA agent is introduced into the body ofa subject. Methods can be used to determine sites of cleavage, e.g.,endo- and exonucleolytic cleavage on an iRNA agent and to determine themechanism of cleavage. An iRNA agent can be modified to inhibit suchcleavage.

A dsRNA, e.g., an iRNA agent, can be evaluated to identify sites thatare susceptible to modification, particularly cleavage, e.g., cleavageby a component found in the body of a subject. The component can bespecific for a particular area of the body, such as a particular tissue,organ, or bodily fluid (e.g., blood, plasma, or serum). Sites in an iRNAagent that are susceptible to cleavage, either by endonucleolytic orexonucleolytic cleavage, in certain areas of the body, may be resistantto cleavage in other areas of the body.

A method for evaluating an iRNA agent can include: (1) determining thepoint or points at which a substance present in the body of a subject,and preferably a component present in a compartment of the body intowhich a therapeutic dsRNA is to be introduced (this includescompartments into which the therapeutic is directly introduced, e.g.,the circulation, as well as in compartments to which the therapeutic iseventually targeted, e.g, the liver or kidney; in some cases, e.g, theeye, the two are the same), cleaves a dsRNA, e.g., an iRNA agent; and(2) identifying one or more points of cleavage, e.g., endonucleolytic,exonucleolytic, or both, in the dsRNA. Optionally, the method furtherincludes providing an RNA (e.g., an iRNA agent) modified to inhibitcleavage at such sites.

The steps described above can be accomplished by using one or more ofthe following assays:

(i) (a) contacting a candidate dsRNA, e.g., an iRNA agent, with a testagent (e.g., a biological agent),

-   -   (b) using a size-based assay, e.g., gel electrophoresis to        determine if the iRNA agent is cleaved. In a preferred        embodiment a time course is taken and a number of samples        incubated for different times are applied to the size-based        assay. In preferred embodiments, the candidate dsRNA is not        labeled. The method can be a “stains all” method.

(ii) (a) supplying a candidate dsRNA, e.g., an iRNA agent, which isradiolabeled;

-   -   (b) contacting the candidate dsRNA with a test agent,    -   (c) using a size-based assay, e.g., gel electrophoresis to        determine if the iRNA agent is cleaved. In a preferred        embodiment, a time course is taken where a number of samples are        incubated for different times and applied to the size-based        assay. In preferred embodiments the determination is made under        conditions that allow determination of the number of nucleotides        present in a fragment. For example, an incubated sample is run        on a gel having markers that allow assignment of the length of        cleavage products. The gel can include a standard that is a        “ladder” digestion. Either the sense or antisense strand can be        labeled. Preferably only one strand is labeled in a particular        experiment. The label can be incorporated at the 5′ end, 3′ end,        or at an internal position. Length of a fragment (and thus the        point of cleavage) can be determined from the size of the        fragment based on the ladder and mapping using a site-specific        endonuclease such as RNAse T1.

(iii) Fragments produced by any method, e.g., one described herein,e.g., one of those above, can be analyzed by mass spectrometry.Following contacting the iRNA with the test agent, the iRNA can bepurified (e.g., partially purified), such as by phenol-chloroformextraction followed by precipitation. Liquid chromatography can then beused to separate the fragments and mass spectrometry can be used todetermine the mass of each fragment. This allows determination of themechanism of cleavage, e.g., if by direct phosphate cleavage, such as by5′ or 3′ exonuclease cleavage, or mediated by the 2′OH via formation ofa cyclic phosphate.

In another embodiment, the information relating to a site of cleavage isused to select a backbone atom, a sugar or a base, for modification,e.g., a modification to decrease cleavage.

Exemplary modifications include modifications that inhibitendonucleolytic degradation, including the modifications describedherein. Particularly favored modifications include: 2′ modification,e.g., a 2′-O-methylated nucleotide or 2′-deoxy nucleotide (e.g., 2′deoxy-cytodine), or a 2′-fluoro, difluorotoluoyl, 5-Me-2′-pyrimidines,5-allyamino-pyrimidines, 2′-β-methoxyethyl, 2′-hydroxy, or 2′-ara-fluoronucleotide, or a locked nucleic acid (LNA), extended nucleic acid (ENA),hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA). In oneembodiment, the 2′ modification is on the uridine of at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide, at least one5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, or at least one5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, or on the cytidine of atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, at least one5′-cytidine-cytidine-3′ (5′-CC-3′) dinucleotide, or at least one5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide. The 2′ modification canalso be applied to all the pyrimidines in an iRNA agent. In onepreferred embodiment, the 2′ modification is a 2′OMe modification on thesense strand of an iRNA agent. In a more preferred embodiment the 2′modification is a 2′ fluoro modification, and the 2′ fluoro is on thesense or antisense strand or on both strands.

Modification of the backbone, e.g., with the replacement of an O with anS, in the phosphate backbone, e.g., the provision of a phosphorothioatemodification can be used to inhibit endonuclease activity. In someembodiments, an iRNA agent has been modified by replacing one or moreribonucleotides with deoxyribonucleotides. Preferably, adjacentdeoxyribonucleotides are joined by phosphorothioate linkages, and theiRNA agent does not include more than four consecutivedeoxyribonucleotides on the sense or the antisense strands. Replacementof the U with a C5 amino linker; replacement of an A with a G (sequencechanges are preferred to be located on the sense strand and not theantisense strand); or modification of the sugar at the 2′, 6′, 7′, or 8′position can also inhibit endonuclease cleavage of the iRNA agent.Preferred embodiments are those in which one or more of thesemodifications are present on the sense but not the antisense strand, orembodiments where the antisense strand has fewer of such modifications.

Exemplary modifications also include those that inhibit degradation byexonucleases. Examples of modifications that inhibit exonucleolyticdegradation can be found herein. In one embodiment, an iRNA agentincludes a phosphorothioate linkage or P-alkyl modification in thelinkages between one or more of the terminal nucleotides of an iRNAagent. In another embodiment, one or more terminal nucleotides of aniRNA agent include a sugar modification, e.g., a 2′ or 3′ sugarmodification. Exemplary sugar modifications include, for example, a2′-β-methylated nucleotide, 2′-deoxy nucleotide (e.g., deoxy-cytodine),2′-deoxy-2′-fluoro (2′-F) nucleotide, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O—N-methylacetamido (2′-O-NMA),2′-O-dimethylaminoethlyoxyethyl (2′-DMAEOE), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-AP), 2′-hydroxy nucleotide,or a 2′-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extendednucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleicacid (CeNA). A 2′ modification is preferably 2′OMe, more preferably, 2′fluoro.

The modifications described to inhibit exonucleolytic cleavage can becombined onto a single iRNA agent. For example, in one embodiment, atleast one terminal nucleotide of an iRNA agent has a phosphorothioatelinkage and a 2′ sugar modification, e.g., a 2′F or 2′OMe modification.In another embodiment, at least one terminal nucleotide of an iRNA agenthas a 5′ Me-pyrimidine and a 2′ sugar modification, e.g., a 2′F or 2′OMemodification.

To inhibit exonuclease cleavage, an iRNA agent can include a nucleobasemodification, such as a cationic modification, such as a 3′-abasiccationic modification. The cationic modification can be, e.g., analkylamino-dT (e.g., a C6 amino-dT), an allylamino conjugate, apyrrolidine conjugate, a pthalamido or a hydroxyprolinol conjugate, onone or more of the terminal nucleotides of the iRNA agent. Analkylamino-dT conjugate is preferably attached to the 3′ end of thesense or antisense strand of an iRNA agent. A pyrrolidine linker ispreferably attached to the 3′ or 5′ end of the sense strand, or the 3′end of the antisense strand. An allyl amine uridine is preferably on the3′ or 5′ end of the sense strand, and not on the 5′ end of the antisensestrand.

In another embodiment, the iRNA agent includes a conjugate on one ormore of the terminal nucleotides of the iRNA agent. The conjugate canbe, for example, a lipophile, a terpene, a protein binding agent, avitamin, a carbohydrate, a retiniod, or a peptide. For example, theconjugate can be naproxen, nitroindole (or another conjugate thatcontributes to stacking interactions), folate, ibuprofen, cholesterol,retinoids, PEG, or a C5 pyrimidine linker. In other embodiments, theconjugates are glyceride lipid conjugates (e.g. a dialkyl glyceridederivatives), vitamin E conjugates, or thio-cholesterols. Preferably,conjugates are on the 3′ end of the antisense strand, or on the 5′ or 3′end of the sense strand, and preferably the conjugates are not on the 3′end of the antisense strand and on the 3′ end of the sense strand.

In one embodiment, the conjugate is naproxen, and the conjugate ispreferably on the 5′ or 3′ end of the sense or antisense strands. In oneembodiment, the conjugate is cholesterol, and the conjugate ispreferably on the 5′ or 3′ end of the sense strand and preferably notpresent on the antisense strand. In some embodiments, the cholesterol isconjugated to the iRNA agent by a pyrrolidine linker, or serinol linker,aminooxy, or hydroxyprolinol linker. In other embodiments, the conjugateis a dU-cholesterol, or cholesterol is conjugated to the iRNA agent by adisulfide linkage. In another embodiment, the conjugate is cholanicacid, and the cholanic acid is attached to the 5′ or 3′ end of the sensestrand, or the 3′ end of the antisense strand. In one embodiment, thecholanic acid is attached to the 3′ end of the sense strand and the 3′end of the antisense strand. In another embodiment, the conjugate isPEGS, PEG20, naproxen or retinal.

In another embodiment, one or more terminal nucleotides have a 2′-5′linkage. Preferably, a 2′-5′ linkage occurs on the sense strand, e.g.,the 5′ end of the sense strand.

In one embodiment, the iRNA agent includes an L-sugar, preferably at the5′ or 3′ end of the sense strand.

In one embodiment, the iRNA agent includes a methylphosphonate at one ormore terminal nucleotides to enhance exonuclease resistance, e.g., atthe 3′ end of the sense or antisense strands of the iRNA agent.

In one embodiment, an iRNA agent has been modified by replacing one ormore ribonucleotides with deoxyribonucleotides. Preferably, adjacentdeoxyribonucleotides are joined by phosphorothioate linkages, and theiRNA agent does not include more than four consecutivedeoxyribonucleotides on the sense or the antisense strands.

In some embodiments, an iRNA agent having increased stability in cellsand biological samples includes a difluorotoluoyl (DFT) modification,e.g., 2,4-difluorotoluoyl uracil, or a guanidine to inosinesubstitution.

The methods described can be used to select and/or optimize atherapeutic dsRNA, e.g., iRNA agent. dsRNAs, e.g., iRNA agents, made bya method described herein are within the invention.

The methods can be used to evaluate a candidate dsRNA, e.g., a candidateiRNA agent, which is unmodified or which includes a modification, e.g.,a modification that inhibits degradation, targets the dsRNA molecule, ormodulates hybridization. Such modifications are described herein. Acleavage assay can be combined with an assay to determine the ability ofa modified or non-modified candidate to silence the target. For example,one might (optionally) test a candidate to evaluate its ability tosilence a target (or off-target sequence), evaluate its susceptibilityto cleavage, modify it (e.g., as described herein, e.g., to inhibitdegradation) to produce a modified candidate, and test the modifiedcandidate for one or both of the ability to silence and the ability toresist degradation. The procedure can be repeated. Modifications can beintroduced one at a time or in groups. It will often be convenient touse a cell-based method to monitor the ability to silence a target RNA.This can be followed by a different method, e.g, a whole animal method,to confirm activity.

The invention includes using information on cleavage sites obtained by amethod described herein to modify a dsRNA, e.g., an iRNA agent.

Optimizing the Activity of the Nucleic Acid Molecules of the Invention

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., Nature344:565, 1990; Phieken et al., Science 253:314, 1991; Usman andCedergren, Trends in Biochem. Sci. 17:334, 1992; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

Other suitable modifications to a sugar, base, or backbone of an iRNAagent are described elsewhere herein.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O— allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, Trends in Biochem. Sci. 17:34, 1992;Usman et al., Nucleic Acids Symp. Ser. 31:163, 1994; Burgin et al.,Biochemistry 35:14090, 1996). Sugar modification of nucleic acidmolecules have been extensively described in the art (see Eckstein etal., International Publication PCT No. WO 92/07065; Perrault et al.,Nature 344:565, 1990; Pieken et al. Science 253:314, 1991; Usman andCedergren, Trends in Biochem. Sci. 17:334, 1992; Usman et al.,International Publication PCT No. WO93/15187; Sproat, U.S. Pat. No.5,334,711, and Beigelman et al., J. Biol. Chem. 270:25702, 1995;Beigelman et al., International PCT publication No. WO 97/26270;Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.5,627,053; Woolf et al., International PCT Publication No. WO 98/13526;Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait,Biopolymers (Nucleic Acid Sciences) 48:39, 1998; Verma and Eckstein,Annu. Rev. Biochem. 67:99, 1998; and Burlina et al., Bioorg. Med. Chem.5:1999, 1997; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the iRNA nucleic acid molecules ofthe instant invention so long as the ability of iRNA agents to promoteRNAi in cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

The 3′ and 5′ ends of an iRNA agent can be modified. Such modificationscan be at the 3′ end, 5′ end or both ends of the molecule. They caninclude modification or replacement of an entire terminal phosphate orof one or more of the atoms of the phosphate group. For example, the 3′and 5′ ends of an oligonucleotide can be conjugated to other functionalmolecular entities such as labeling moieties, e.g., fluorophores (e.g.,pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (basede.g., on sulfur, silicon, boron or ester). The functional molecularentities can be attached to the sugar through a phosphate group and/or aspacer. The terminal atom of the spacer can connect to or replace thelinking atom of the phosphate group or the C-3′ or C-5′ O, N, S or Cgroup of the sugar. Alternatively, the spacer can connect to or replacethe terminal atom of a nucleotide surrogate (e.g., PNAs). These spacersor linkers can include e.g., —(CH2)n—, —(CH2)nN—, —(CH2)nO—, —(CH2)nS—,O(CH2CH2O)nCH2CH2OH (e.g., n=3 or 6), abasic sugars, amide, carboxy,amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide,or morpholino, or biotin and fluorescein reagents. When aspacer/phosphate-functional molecular entity-spacer/phosphate array isinterposed between two sequences of an iRNA agent, the array cansubstitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′end can be an —OH group. While not wishing to be bound by theory, it isbelieved that conjugation of certain moieties can improve transport,hybridization, and specificity properties. Again, while not wishing tobe bound by theory, it may be desirable to introduce terminalalterations that improve nuclease resistance. Other examples of terminalmodifications include dyes, intercalating agents (e.g., acridines),cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA),lipophilic carriers (e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.,biotin), transport/absorption facilitators (e.g., aspirin, vitamin E,folic acid), and synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles). In some embodiments, conjugates suchas retinol or retinoic acid can be attached to the 5′ or 3′ end, or bothends, of an iRNA agent. Use of such conjugates may improve specificuptake and delivery of iRNA agents to cells that express retinolreceptors, such as retinal pigment epithelial cells.

Terminal modifications can be added for a number of reasons, such as tomodulate activity or to modulate resistance to degradation. Terminalmodifications useful for modulating activity include modification of the5′ end with phosphate or phosphate analogs. For example, in preferredembodiments iRNA agents, especially antisense sequences, are 5′phosphorylated or include a phosphoryl analog at the 5′ prime terminus.5′-phosphate modifications include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P-β-(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.,RP(OH)(O)—O-5′-).

In another embodiment, the invention features conjugates and/orcomplexes of iRNA agents of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of iRNA agents into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,phospholipids, nucleosides, nucleotides, nucleic acids, antibodies,toxins, negatively charged polymers and other polymers, for example,proteins, peptides, hormones, carbohydrates, polyethylene glycols, orpolyamines, across cellular membranes. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system, with or without degradable linkers. Thesecompounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

Administration of the iRNA Agents

A patient who has been diagnosed with a disorder characterized byunwanted VEGF expression can be treated by administration of an iRNAagent described herein to block the negative effects of VEGF, therebyalleviating the symptoms associated with unwanted VEGF gene expression.For example, the iRNA agent can alleviate symptoms associated with adisease of the eye, such as a neovascular disorder. In other examples,the iRNA agent can be administered to treat a patient who has a tumor ormetastatic cancer, such as colon or breast cancer; a pulmonary disease,such as asthma or bronchitis; or an autoimmune disease such asrheumatoid arthritis or psoriasis. The anti-VEGF iRNA agents can beadministered systemically, e.g., orally or by intramuscular injection orby intravenous injection, in admixture with a pharmaceuticallyacceptable carrier adapted for the route of administration. An iRNAagent can comprise a delivery vehicle, including liposomes, foradministration to a subject, carriers and diluents and their salts,and/or can be present in pharmaceutically acceptable formulations.Methods for the delivery of nucleic acid molecules are described inAkhtar et al., Trends in Cell Bio. 2:139, 1992; Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed Akhtar, 1995; Maurer et al.,Mol. Membr. Biol., 16:129, 1999; Hofland and Huang, Handb. Exp.Pharmacol. 137:165, 1999; and Lee et al., ACS Symp. Ser. 752:184, 2000,all of which are incorporated herein by reference. Beigelman et al.,U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 furtherdescribe the general methods for delivery of nucleic acid molecules.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by ionophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins (seefor example Gonzalez et al., Bioconjugate Chem. 10:1068, 1999),biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (O'Hare and Normand, International PCT PublicationNo. WO 00/53722).

In the present methods, the iRNA agent can be administered to thesubject either as naked iRNA agent, in conjunction with a deliveryreagent, or as a recombinant plasmid or viral vector which expresses theiRNA agent. Preferably, the iRNA agent is administered as naked iRNA.

The iRNA agent of the invention can be administered to the subject byany means suitable for delivering the iRNA agent to the cells of thetissue at or near the area of unwanted VEGF expression, such as at ornear an area of neovascularization. For example, the iRNA agent can beadministered by gene gun, electroporation, or by other suitableparenteral administration routes.

Suitable enteral administration routes include oral delivery.

Suitable parenteral administration routes include intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., intraocular injection, intra-retinal injection, or sub-retinalinjection); subcutaneous injection or deposition including subcutaneousinfusion (such as by osmotic pumps); direct application to the area ator near the site of neovascularization, for example by a catheter orother placement device (e.g., a retinal pellet or an implant comprisinga porous, non-porous, or gelatinous material). It is preferred thatinjections or infusions of the iRNA agent be given at or near the siteof neovascularization.

The iRNA agent of the invention can be delivered using an intraocularimplant. Such implants can be biodegradable and/or biocompatibleimplants, or may be non-biodegradable implants. The implants may bepermeable or impermeable to the active agent, and may be inserted into achamber of the eye, such as the anterior or posterior chambers, or maybe implanted in the sclera, transchoroidal space, or an avascularizedregion exterior to the vitreous. In a preferred embodiment, the implantmay be positioned over an avascular region, such as on the sclera, so asto allow for transcleral diffusion of the drug to the desired site oftreatment, e.g., the intraocular space and macula of the eye.Furthermore, the site of transcleral diffusion is preferably inproximity to the macula.

The iRNA agent of the invention can also be administered topically, forexample, by patch or by direct application to the eye, or byiontophoresis. Ointments, sprays, or droppable liquids can be deliveredby ocular delivery systems known in the art such as applicators oreyedroppers. The compositions can be administered directly to thesurface of the eye or to the interior of the eyelid. Such compositionscan include mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers.

The iRNA agent of the invention may be provided in sustained releasecompositions, such as those described in, for example, U.S. Pat. Nos.5,672,659 and 5,595,760. The use of immediate or sustained releasecompositions depends on the nature of the condition being treated. Ifthe condition consists of an acute or over-acute disorder, treatmentwith an immediate release form will be preferred over a prolongedrelease composition. Alternatively, for certain preventative orlong-term treatments, a sustained release composition may beappropriate.

An iRNA agent can be injected into the interior of the eye, such as witha needle or other delivery device.

The iRNA agent of the invention can be administered in a single dose orin multiple doses. Where the administration of the iRNA agent of theinvention is by infusion, the infusion can be a single sustained dose orcan be delivered by multiple infusions. Injection of the agent directlyinto the tissue is at or near the site of neovascularization ispreferred. Multiple injections of the agent into the tissue at or nearthe site of neovascularization are also preferred.

Dosage levels on the order of about 1 μg/kg to 100 mg/kg of body weightper administration are useful in the treatment of the neovasculardiseases. When administered directly to the eye, the preferred dosagerange is about 0.00001 mg to about 3 mg per eye, or preferrably about0.0001-0.001 mg per eye, about 0.03-3.0 mg per eye, about 0.1-3.0 mg pereye or about 0.3-3.0 mg per eye. One skilled in the art can also readilydetermine an appropriate dosage regimen for administering the iRNA agentof the invention to a given subject. For example, the iRNA agent can beadministered to the subject once, e.g., as a single injection ordeposition at or near the neovascularization site. Alternatively, theiRNA agent can be administered once or twice daily to a subject for aperiod of from about three to about twenty-eight days, more preferablyfrom about seven to about ten days. In a preferred dosage regimen, theiRNA agent is injected at or near a site of unwanted VEGF expression(such as near a site of neovascularization) once a day for seven days.Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of iRNA agent administered to thesubject can comprise the total amount of iRNA agent administered overthe entire dosage regimen. One skilled in the art will appreciate thatthe exact individual dosages may be adjusted somewhat depending on avariety of factors, including the specific iRNA agent beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisorder being treated, the severity of the disorder, thepharmacodynamics of the iRNA agent, and the age, sex, weight, andgeneral health of the patient. Wide variations in the necessary dosagelevel are to be expected in view of the differing efficiencies of thevarious routes of administration. For instance, oral administrationgenerally would be expected to require higher dosage levels thanadministration by intravenous or intravitreal injection. Variations inthese dosage levels can be adjusted using standard empirical routines ofoptimization, which are well-known in the art. The precisetherapeutically effective dosage levels and patterns are preferablydetermined by the attending physician in consideration of theabove-identified factors.

In addition to treating pre-existing neovascular diseases, iRNA agentsof the invention can be administered prophylactically in order toprevent or slow the onset of these and related disorders. Inprophylactic applications, an iRNA of the invention is administered to apatient susceptible to or otherwise at risk of a particular neovasculardisorder.

The iRNA agents featured by the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. As used herein, “pharmaceutical formulations” includeformulations for human and veterinary use. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example as described in Remington's Pharmaceutical Science,18th ed., Mack Publishing Company, Easton, Pa. (1990), and The Scienceand Practice of Pharmacy, 2003, Gennaro et al., the entire disclosuresof which are herein incorporated by reference.

The present pharmaceutical formulations comprise an iRNA agent of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and thelike.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For solid compositions, conventional non-toxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more iRNA agents of the invention.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as PluronicP85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, Fundam. Clin. Pharmacol. 13:16, 1999); biodegradablepolymers, such as poly(DL-lactide-coglycolide) microspheres forsustained release delivery. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., J. Pharm. Sci. 87:1308,1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al., PNASUSA. 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995;Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; and Tyler etal., PNAS USA 96:7053, 1999.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly(ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al., Chem. Rev.95:2601, 1995; Ishiwata et al., Chem. Phare. Bull. 43:1005, 1995).

Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 267:1275, 1995; Oku et al., Biochim.Biophys. Acta 1238:86, 1995). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864,1995; Choi et al., International PCT Publication No. WO 96/10391; Ansellet al., International PCT Publication No. WO 96/10390; Holland et al.,International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

Alternatively, certain iRNA agents of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, Science 229:345, 1985; McGarry and Lindquist, Proc. Natl.Acad. Sci. USA 83:399, 1986; Scanlon et al., Proc. Natl. Acad. Sci. USA88:10591, 1991; Kashani-Sabet et al., Antisense Res. Dev. 2:3, 1992;Dropulic et al., J. Virol. 66:1432, 1992; Weerasinghe et al., J. Virol.65:5531, 1991; Ojwang et al., Proc. Natl. Acad. Sci. USA 89:10802, 1992;Chen et al., Nucleic Acids Res. 20:4581, 1992; Sarver et al., Science247:1222, 1990; Thompson et al., Nucleic Acids Res. 23:2259, 1995; Goodet al., Gene Therapy 4:45, 1997). Those skilled in the art realize thatany nucleic acid can be expressed in eukaryotic cells from theappropriate DNA/RNA vector. The activity of such nucleic acids can beaugmented by their release from the primary transcript by a enzymaticnucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCTWO 94/02595; Ohkawa et al., Nucleic Acids Symp. Ser. 27:156, 1992; Tairaet al., Nucleic Acids Res. 19:5125, 1991; Ventura et al., Nucleic AcidsRes. 21:3249, 1993; Chowrira et al., J. Biol. Chem. 269:25856, 1994).

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., Trends in Genetics 12:510, 1996) inserted into DNA orRNA vectors. The recombinant vectors can be DNA plasmids or viralvectors. iRNA agent-expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus, retrovirus, adenovirus,or alphavirus. In another embodiment, pol III based constructs are usedto express nucleic acid molecules of the invention (see for exampleThompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinantvectors capable of expressing the iRNA agents can be delivered asdescribed above, and persist in target cells. Alternatively, viralvectors can be used that provide for transient expression of nucleicacid molecules. Such vectors can be repeatedly administered asnecessary. Once expressed, the iRNA agent interacts with the target mRNAand generates an RNAi response. Delivery of iRNA agent-expressingvectors can be systemic, such as by intravenous or intra-muscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell(for a review see Couture et al., Trends in Genetics 12:510, 1996).

Additional ophthalmic indications for the iRNA agents of the inventioninclude proliferative diabetic retinopathy (the most severe stage ofdiabetic retinopathy), uveitis (an inflammatory condition of the eyethat often leads to macular edema), cystoid macular edema followingcataract surgery, myopic degeneration (a condition in which a patientwith a high degree of nearsightedness develops choroidalneovascularization), inflammatory macular degeneration (a condition inwhich a patient with inflammation in the macular area due to infectionsor other causes, develops choroidal neovascularization), and irisneovascularization (a serious complication of diabetic retinopathy orretinal vein occlusion involving new blood vessel growth on the surfaceof the iris).

Additional non-ophthalmic indications for the iRNA agents of theinvention include cancer, including but not limited to renal and coloncancer, and psoriasis. Solid tumors and their metastases rely on newblood vessel growth for their survival.

Psoriasis is a chronic inflammatory skin disease that causes skin cellsto grow too quickly, resulting in thick white or red patches of skin.Preclinical and clinical data suggest that VEGF-induced blood vesselgrowth and blood vessel leakage play a role in the development of thiscondition.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 siRNA Design

Four hundred target sequences were identified within exons 1-5 of theVEGF-A121 mRNA sequence (See Table 1, SEQ ID NOs 2-401) andcorresponding siRNAs targeting these subjected to a bioinformaticsscreen.

To ensure that the sequences were specific to VEGF sequence and not tosequences from any other genes, the target sequences were checkedagainst the sequences in Genbank using the BLAST search engine providedby NCBI. The use of the BLAST algorithm is described in Altschul et al.,J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol.266:460, 1996.

siRNAs were also prioritized for their ability to cross react withmonkey, rat and human VEGF sequences.

Of these 400 potential target sequences 80 were selected for analysis byexperimental screening in order to identify a small number of leadcandidates. A total of 114 siRNA molecules were designed for these 80target sequences 114 (Table 2).

Example 2 Synthesis of the siRNA Oligonucleotides

RNA was synthesized on Expedite 8909, ABI 392 and ABI394 Synthesizers(Applied Biosystems, Applera Deutschland GmbH, Frankfurter Str. 129b,64293 Darmstadt, Germany) at 1 mmole scale employing CPG solid supportand Expedite RNA phosphoramidites (both from Proligo Biochemie GmbH,Georg-Hyken-Str.14, Hamburg, Germany). Ancillary reagents were obtainedfrom Mallinckrodt Baker (Im Leuschnerpark 4:64347 Griesheim, Germany).Phosphorothioate linkages were introduced by replacement of the iodineoxidizer solution with a solution of the Beaucage reagent inacetonitrile (5% weight per volume).

Cleavage of the oligoribonucleotides from the solid support and basedeprotection was accomplished with a 3:1 (v/v) mixture of methylamine(41%) in water and methylamine (33%) in ethanol. 2′-Desilylation wascarried out according to established procedures (Wincott et al., NucleicAcids Res. 23:2677-2684, 1995). Crude oligoribonucleotides were purifiedby anion exchange HPLC using a 22×250 mm DNAPac PA 100 column withbuffer A containing 10 mM NaClO₄, 20 mM Tris, pH 6.8, 6 M urea andbuffer B containing 400 mM NaClO₄, 20 mM Tris, pH 6.8, 6 M Urea. Flowrate was 4.5 mL/min starting with 15% Buffer B which was increased to55% over 45 minutes.

The purified compounds were characterized by LC/ESI-MS (LC: Ettan Micro,Amersham Biosciences Europe GmbH, Munzinger Strasse 9, 79111 Freiburg,Germany, ESI-MS: LCQ, Deca XP, Thermo Finnigan, Im Steingrund 4-6, 63303Dreieich, Germany) and capillary electrophoresis (P/ACE MDQ CapillaryElectrophoresis System, Beckman Coulter GmbH, 85702 Unterschleiβheim,Germany). Purity of the isolated oligoribonucleotides was at least 85%.

Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer. Double stranded RNA was generated by mixing an equimolarsolution of complementary strands in annealing buffer (20 mM sodiumphosphate, pH 6.8; 100 mM sodium chloride), heating in a water bath at85-90° C. for 3 minutes and cooling to room temperature over a period of3-4 hours. The RNA was kept at −20° C. until use.

Example 3 Efficacy Screen of siRNAs

Using two efficacy screens, the VEGF siRNA were screened for theirability to become a lead candidate. Table 2 shows the relativeefficiencies of some of the siRNAs in their ability to inhibitexpression of an endogenous VEGF gene. In this process the number ofcandidate siRNAs was winnowed. Human HeLa or ARPE-19 (human retinalpigment epithelial cell line with differentiated properties (Dunn etal., Exp. Eye Res. 62:155, 1996) were plated in 96-well plates (17,000cells/well) in 100 μl 10% fetal bovine serum in Dulbecco's ModifiedEagle Medium (DMEM). When the cells reached approximately 90% confluence(approximately 24 hours later) they were transfected with serialthree-fold dilutions of siRNA starting at 20 nM 0.4 μl of transfectionreagent Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, Calif.)was used per well and transfections were performed according to themanufacturer's protocol. Namely, the siRNA: Lipofectamine™ 2000complexes were prepared as follows. The appropriate amount of siRNA wasdiluted in Opti-MEM I Reduced Serum Medium without serum and mixedgently. The Lipofectamine™ 2000 was mixed gently before use, then foreach well of a 96 well plate, 0.4 μl was diluted in 25 μl of Opti-MEM IReduced Serum Medium without serum and mixed gently and incubated for 5minutes at room temperature. After the 5 minute incubation, 1 μl of thediluted siRNA was combined with the diluted Lipofectamine™2000 (totalvolume is 26.4 μl). The complex was mixed gently and incubated for 20minutes at room temperature to allow the siRNA: Lipofectamine™ 2000complexes to form. Then 100 μl of 10% fetal bovine serum in DMEM wasadded to each of the siRNA:Lipofectamine™ 2000 complexes and mixedgently by rocking the plate back and forth. 100 μl of the above mixturewas added to each well containing the cells and the plates wereincubated at 37° C. in a CO₂ incubator for 24 hours, then the culturemedium was removed and 100 μl 10% fetal bovine serum in DMEM was added.Following the medium change, conditioned medium was collected at 24hours (HeLa cells) or 72 hours (ARPE-19 cells) and a human VEGF ELISAwas performed using the DuoSet human VEGF ELISA Development kit (R&DSystems, Inc. Minneapolis, Minn. 55413). This kit contains the basiccomponent required for the development of sandwich ELISAs to measurenatural and recombinant human VEGF in cell culture supernatants andserum.

The materials used included:

Capture Antibody—576 μg/ml of goat anti-human VEGF when reconstitutedwith 0.25 ml PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mMKH₂PO₄, pH 7.2-7.4, 0.2 μm filtered). After reconstitution, stored at2-8° C. for up to 60 days or aliquoted and stored at −20° C. to −70° C.in a manual defrost freezer for up to 6 months. Diluted to a workingconcentration of 0.8 μg/ml in PBS without carrier protein.

Detection antibody—4.5 μg/ml of biotinylated goat anti-human VEGF whenreconstituted with 1.0 ml of Reagent Diluent (1% bovine serum albumin inPBS, pH 7.2-7.4, 0.2 μm filtered. After reconstitution, stored at 2-8°C. for up to 60 days or aliquoted and stored at −20° C. to −70° C. in amanual defrost freezer for up to 6 months. Diluted to a workingconcentration of 25 ng/ml in Reagent Diluent.

Standard: 110 ng/ml of recombinant when reconstituted with 0.5 ml ofReagent Diluent. Allowed the standard to sit for a minimum of 15 minuteswith gentle agitation prior to making dilutions. The reconstitutedStandard can be stored at 2-8° C. for up to 60 days or aliquoted andstored at −20° C. to −70° C. in a manual defrost freezer for up to 6months. A seven point standard curve using 2-fold serial dilutions inReagent Diluent, and a high standard of 4000 pg/ml is recommended.

Streptavidin-HRP: 1.0 ml of streptavidin conjugated tohorseradish-peroxidase. Stored at 2-8° C. for up to 6 months. Diluted tothe working concentration specified on the vial label.

General ELISA protocol was followed (R&D Systems, Inc., Minneapolis,Minn.).

Controls included no siRNA, human VEGF siRNA (Candy, (a.k.a., hVEGF5)Reich et al., Mol. Vis. 9:210, 2003) and an siRNA matching a 21-ntsequence conserved between the human, rat and mouse VEGF (hrmVEGF,Filleur et al., Cancer Res. 63:3919-3922, 2003).

The activities of the siRNAs were compared to the activity of thecontrol human VEGF siRNA of Reich et al. (supra) with “+” representing alower activity, “++” representing similar activity and “+++”representing a higher activity than the control human VEGF siRNA (Table2). FIG. 2 shows the activities of single- and double-overhang siRNAs inHeLa cells. Solid lines with filled symbols represent thesingle-overhang siRNA, solid lines with open symbols represent thedouble-overhang siRNAs; dashed lines represent the control siRNAs. Allof the siRNAs are more active than the control siRNAs and may inhibitexpression of VEGF by approximately 80%. In contrast, the siRNA fromReich et al. (supra) reduced the level of endogenous hVEGF byapproximately 20% under the same experimental conditions. Similarly,under the same experimental conditions, the siRNA based on consensussequence hrmVEGF (Filleur et al., supra) reduced the expression level byapproximately 45%.

FIG. 3 shows the activities of single- and double-overhang siRNAs inARPE-19 cells. Solid lines with filled symbols represent thesingle-overhang siRNA, solid lines with open symbols represent thedouble-overhang siRNAs; dashed lines represent the control siRNAs. Allof the siRNAs are more active than the control siRNAs and may inhibitexpression of VEGF by approximately 90%. In contrast, the siRNA fromReich et al. (Mol. Vis. 9:210, 2003) reduced the level of hVEGF byapproximately 35% under the same experimental conditions. Similarly,under the same experimental conditions, the siRNA based on consensussequence hrmVEGF (Filleur et al., supra) reduced the expression level byapproximately 70%.

FIGS. 4 and 5 show the results of a comparison of single- anddouble-overhang siRNAs with their analogous blunt-ended siRNAs,respectively in HeLa cells. The results are in agreement with the dataof Elbashir et al. (Genes & Development 15:188, 2001) in that thepresence of an overhang in an siRNA confers greater efficiency ininhibition of gene silencing. However, it is important to note that theactivity of the blunt ended siRNAs are comparable to the resultsachieved using the control siRNAs.

Example 4 In Vitro Assay for the Silencing of VEGF Synthesis UnderHypoxic Conditions

Human HeLa cells were plated in 96 well plates at 10,000 cells/well in100 μl of growth medium (10% FBS in DMEM). 24 hours post cell seedingwhen the cells had reached approximately 50% confluence they weretransfected with serial three fold dilutions of siRNA starting at 30 nM.0.2 μl of Lipofectamine™ 2000 transfection reagent (InvitrogenCorporation, Carlsbad Calif.) was used per well and transfections werecarried out as described in the Invitrogen product insert. Controlsincluded no siRNA, human VEGF siRNA (Reich et al., Mol. Vis. 9:210,2003) and an siRNA matching a 21-nt sequence conserved between thehuman, rat and mouse VEGF (hrmVEGF, Filleur et al., Cancer Res.63:3919-3922, 2003). Transfections were done in duplicate on each plate.Additionally, duplicate plates were transfected so that 24 hours posttransfection the growth media could be changed and one plate could bekept in normal oxygen growth conditions (37° C., 5% CO₂, 20% oxygen) andthe duplicate plate could be kept in hypoxic conditions (37° C., 1%oxygen, balance nitrogen). Hypoxic conditions were maintained by using aPro-ox Oxygen Controller (BioSpherix, Ltd., Redfield, N.Y.) attached toa Pro-ox in vitro culture chamber. Cells were maintained in eithernormoxic or hypoxic conditions for 24 hours post media change.Conditioned culture media was then collected from both plates and testedfor secreted VEGF levels in a DuoSet VEGF ELISA (R&D Systems,Minneapolis, Minn.). The assays were performed according to themanufacturer's protocol and as described in Example 2.

For deferoxamine chemically induced hypoxia, 130 μM deferoxamine (SigmaD9533), was used. Deferoxamine was added to the fresh growth media 24hours post-transfection. Cells treated with deferoxamine were then grownunder normal growth conditions (37° C., 5% CO₂, 20% oxygen).

FIG. 6 shows the results obtained with siRNAs (both single overhangsiRNAs and double overhangs siRNAs) directed against ORF regions havingthe first nucleotides corresponding to 319 and 343 respectively,together with the control siRNAs. Under hypoxic conditions, either 1%oxygen (FIG. 6B) or 130 μM deferoxamine (FIG. 6C), three of theexperimental siRNAs achieved almost 95% inhibition of expression ofVEGF, namely AL-DP-4094 (single-overhang) directed at ORF 343, and bothof the siRNAs (single and double-overhangs) directed at ORF 319. Thecontrol siRNAs of Reich et at (supra) and Filleur et al. (supra)demonstrate an ability to inhibit VEGF expression by 45% and 85%respectively.

FIGS. 8A and 8B show the results obtained with the siRNAs AL-DP-4014, aphosphorothioate modified version of AL-DP-4014 (AL-DP-4127, see Table3) and a mutated version of AL-DP-4014 (AL-DP-4140, see Table 5). Underboth normal and hypoxic conditions, the unmodified (AL-DP-4014) and thephosphorothioate-modified siRNA (AL-DP-4127) reduced endogenous VEGFexpression to less than 20% of its original expression level. Underhypoxic conditions, the phosphorothioate-modified siRNA essentiallyabolished VEGF expression.

Example 5 Modified VEGF siRNA Molecules Retain Full Activity and ShowEnhanced Stability

Phosphorothioate derivatives were made for the AL-DP-4014, targeting ORF319 of VEGF, and are presented in Table 3. These siRNAs were tested inthe HeLa cell assay described in Example 3, and FIG. 7 shows that thesederivatives are as active in the HeLa assay as the unmodified siRNA.

A panel of siRNAs were synthesized that retained the sequence of theAL-DP-4094 siRNA (Table 1) but included different modificationsincluding phosphorothioate linkages, O-methyl-modified nucleotides, and2′-fluoro-modified nucleotides (Table 4). The panel of siRNAs was testedin HeLa cells, and FIGS. 9A-9E demonstrate that all modified versions ofthe AL-DP-4094 siRNA effectively reduced VEGF expression by greater than90%, exhibiting greater efficacy than either of the two previouslyidentified VEGF siRNAs (“Acuity” in Reich et al. (supra), and “Filleur”in Filleur et al. (supra)). FIG. 10 also shows data from in vitro assaysin HeLa cells. The graph in FIG. 10 shows that the unmodified AL-DP-4094siRNA and a phosphorothioate-modified AL-DP-4004 siRNA (AL-DP-4219)reduced VEGF expression by more than 70% (FIG. 10). Scrambled versionsof the compound AL-DP-4094 (e.g., AL-DP-4216 and AL-DP-4218 (sequencesshown below; underlined nucleotides represent mismatched nucleotides ascompared to AL-DP-4094)) did not inhibit VEGF expression. An siRNAtargeting the firefly luciferase gene (AL-DP-3015; see below) also didnot inhibit VEGF expression.

AL-DP-4216 AL4094 MI s  5′- GCACAUUGGACAGUUGUGGUU-3′ (SEQ ID NO: 1065)AL4094 MI as ′3-GUCGUGUAACCUGUCAACACCAA-′5 (SEQ ID NO: 1091) AL-DP-4218AL4094 M5 s  5′- GCACAUAGAAGUGACGCGCUU-3′ (SEQ ID NO: 1062) AL4094 M5 as′3-GUCGUGUAUCUUCACUGCGCGAA-′5 (SEQ ID NO: 1063) AL-DP-3015 5′- GAACUGUGUGUGAGAGGUCCU-3′ (SEQ ID NO: 830)′3-CGCUUGACACACACUCUCCAGGA-′5 (SEQ ID NO: 831)

The Stains-All technique (Sigma, St. Louis, Mo.) was performed toexamine the stability of the modified siRNAs. To perform the assay, ansiRNA duplex was incubated in 90% human serum at 37° C. Samples of thereaction mix were quenched at various time points (at 0, 0.25, 1, 2, 4,and 24 hours) and subjected to polyacrylamide gel electrophoresis.Cleavage of the RNA over the time course provided information regardingthe susceptibility of the siRNA duplex to serum nuclease degradation.

O-methyl and 2′ fluoro modifications used in combination withphosphorothioate modifications were found to enhance stability to agreater extent than when phosphorothioate modifications were used alone.For example, modified versions of the AL-DP-4094 siRNA included aphosphorothioate-modified siRNA (AL-DP-4198), a phosphorothioate plusO-methyl modified siRNAs (e.g., AL-DP-4180, AL-DP-4175, and AL-DP-4220),and phosphorothioate plus O-methyl plus 2′-fluoro modified siRNAs (e.g.,AL-DP-4197 and AL-DP-4221) (Table 4). The AL-DP-4180, AL-DP-4175, andAL-DP-4197 siRNAs were found to be more stable in human serum than theAL-DP-4198 siRNA. It was determined that the phosphorothioatemodification stabilized the siRNAs against exonucleolytic degradation,and the O-methyl and 2′-fluoro modifications stabilized the siRNAsagainst endonucleolytic degradation.

Example 6 In Vitro Stability Assay of VEGF siRNAs in Different Rat Serumand Ocular Tissues

1. Preparation of Tissue Homogenates

Tissues from pooled whole eyes, retinas, vitreous humors from at leastthree rats were excised and frozen immediately in liquid nitrogen. Thefrozen tissue was pulverized over dry ice, using instruments that werepre-chilled on dry ice. 1 ml of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150mM NaCL, 1 mM Na₂EDTA, 0.5% Na-deoxycholate deoxycholic acid, 1% IGEPALCA-630, 0.05% SDS) was added to the frozen tissue powder and the mixturewas mixed thoroughly and vigorously. The homogenate was centrifuged at10,000×g for 5 min at 4° C. and the pellet was discarded. 100 μlaliquots of the supernatant were transferred to pre-chilledmicrocentrifuge tubes and stored at −70° C. or used immediately in thestability assay.

2. 5′-End Labeling of Single Stranded Sense or Antisense siRNA Using T4Polynucleotide Kinase and γ ³²P-ATP

The following reagents were used:

-   -   T4 Polynucleotide Kinase (PNK) 10 units/μl (New England Biolabs,        Beverly, Mass.)    -   10×T4 PNK buffer (700 mM Tris-HCl, 100 mM MgCl₂, 50 mM        Dithiothreitol (DTT), pH7.6)    -   Gamma-³²P-ATP (PerkinElmer, Shelton, Conn.) 250 μCi, 3000        Ci/mmol (3.3 μM)    -   10 μM stocks of synthetic RNA oligo diluted in H₂O    -   Microspin Sephadex™ G-25 columns (Amersham Biosciences    -   RNAse-free Water and 0.65 ml microcentrifuge (1.5 ml) tubes        A 25 μl kinase reaction contained:    -   2.5 μl from 10 μM stock sense or antisense (1 μM final conc.)    -   2.5 μl 10×PNK Buffer (1×)    -   1.5 μl γ ³²-ATP (approximately 0.2 nM)    -   1.0 μl 10 unit/μl T4 PNK (10 units)    -   17.5 μl dH₂O        The reaction mix was incubated at 37° C. for 1 hour (water bath)        prior to fractionating the labeled siRNA through Sephadex™ G-25        spin columns (Amersham). 0.5 μL was used to determine the number        of counts per minute (cpm)/ml of the radiolabeled sample.

3. Partial Alkaline Hydrolysis Ladder of Radiolabeled Single-StrandedsiRNA

To generate a sample of size markers a portion of the 5′ γ³²P-end-labeled siRNA was subjected to alkaline hydrolysis as follows:

30 μl hydrolysis reaction containing 2.5 μl 5′ end-labeled siRNA (senseor antisense), 6.0 μl 0.5M Na₂CO₃/NaHCO₃ (pH 9.5), 1.5 μl 10 mg/ml tRNA,and 20.0 μl dH₂O was incubated at 90° C. for 7.5 min, then chilled onice or at 4° C. 30 μl of 90% formamide, 50 mM Na₂EDTA, 10 mM DTT, andXC&BB (xylene cyanol and bromophenol blue), of which 1 μl+4 μl formamidedye was used for the gel electrophoretic analysis.

4. Annealing of Radiolabeled 1 μM Stock siRNA Duplexes

30 μl 1 μM stock of different siRNA duplexes were prepared in whicheither the sense strand or the antisense strand was radiolabeled.

The samples were heated at 90° C. for 2 min and then incubated at 37° C.for 1 hour and then stored at −20° C. until used.

5. Quality Control of siRNA Duplex:

Samples of the siRNA duplex were analyzed by electrophoresis through 15%polyacrylamide in Tris-Borate, EDTA (TBE) Gel. Electrophoresis wasperformed at 150V for 1 hour prior to running the samples through.Samples were prepared by mixing 0.5-1 μl siRNA duplex or singlestranded, 3-3.5 μl 0.5×TBE, 1 μl 5× native loading dye (total volume=5μl).

6. Stability Reactions

2 μl siRNA duplex was added to 18 μl serum or tissue lysate or buffercontrol in PCR tube (0.2 ml). A zero time point sample was removedimmediately following the addition of the siRNA duplex by removing 2 μland adding it to 18 μl 90% formamide, 50 mM EDTA, 10 mM DTT and xylenecyanol and bromophenol blue (XC & BB). Other samples were removed after15 min, 30 min, 1 hour, 2 hours, and 4 hours and treated similarly.These samples were stored in a 96-well plate. In some experiments thetime points were extended to 8, 24 and 48 hours. Time point samples forthe buffer (phosphate buffered saline, PBS, 1× working PBS contains 0.14M Sodium Chloride, 0.003 M Potassium Chloride, 0.002 M Potassiumphosphate, 0.01 M Sodium phosphate) were taken at zero and the last timepoint of the experiment. Samples were analyzed by electrophoresisthrough 20% polyacrylamide gels (pre-run at 75 W for 1 hour) in 1×TBE(10 X=890 mM Tris, 890 mM Boric acid, 20 mM EDTA, pH 8.0). The gel wastransferred to a phosphorimager cassette, covered with an enhancerscreen and scanned after overnight exposure.

Polyacrylamide gel analysis indicated that the ocular environmentcontains fewer nucleases than human serum. Testing the unmodified formof the VEGF siRNA AL-DP-4014 for stability in rat eye extract revealedonly the presence of exonuclease activity. In human serum, experimentswith AL-DP-4127 and -4140 (Tables 4 and 5) indicated that terminallymodified phosphorothioate modifications protected against exonucleolyticdegradation but not against endonucleolytic activity. These results wereconsistent with experiments performed in rat whole eye extracts. Theterminally modified phosphorothioate derivatives AL-DP-4127 and -4140were stabilized against exonuclease activity as compared to theunmodified AL-DP-4014 siRNA and the unmodified Cand5 siRNA (Reich et al.(supra)). However, the -4127 and -4140 siRNAs were still subject toendonucleolytic degradation.

Modifications to the lead compound AL-DP-4094 stabilized the siRNAagainst exonucleolytic and endonucleolytic degradation. Thephosphorothioate-modified siRNA AL-DP-4198 was degraded to a similarextent as the unmodified 4094 compound, but the addition of O-methylmodifications, as in AL-DP-4180 and AL-DP-4220, stabilized the siRNAs inrat whole eye extracts.

Notably, the siRNAs were generally more stable in rat retina lysatesthan in the rat whole eye extracts described above. Neither theunmodified AL-DP-4094, nor the modified AL-DP-4198, -4180, or -4220siRNAs were degraded in the retina lysates.

Example 7 Endonuclease-Sensitive Sites were Mapped on AL-DP-4094 siRNA

The stability of the AL-DP-4094 siRNA was examined by the Stains-All andradiolabeled techniques following incubation in human serum (see above).These assays revealed susceptibility to exo- and endonucleases. RP-HPLCwas used to examine the fragment profile of the siRNA followingincubation in serum FIG. 11.

Following incubation of the -4094 siRNA in human serum, the fragmentswere phenol-chloroform extracted and precipitated, and then subjected toLC/MS analysis. FIG. 12 describes the identified fragments andassociated characteristics.

Example 8 Detailed Study of Modifications to siRNAs Targeting VEGF(Table 6)

Eight major different patterns of chemical modification of siRNAduplexes that target the VEGF mRNA were synthesized and evaluated (Table6). The ribose sugar modifications used were either 2′-O-methyl (2′OMe)or 2′-fluoro (2′F). Both pyrimidines (Py) and purines (Pu) could bemodified as provided in Table 6.

The first four patterns (A-D) incorporated 2′OMe on both strands atevery other position. Four configurations were synthesized: 1) at eacheven position on the sense strand and at each odd position of theantisense strand, 2) at each odd position on the sense strand and ateach even position of the antisense strand, 3) at even positions on bothstrands, and 4) at odd positions on both strands.;

The fifth pattern (E) incorporated the 2′OMe modification at allpyrimidine nucleotides on both the sense and antisense strands of theduplex.

Pattern F included duplexes with 2′OMe modifications only on pyrimidinesin 5′-PyPu-3′ dinucleotides, especially at only at UA, CA, UG sites(both strands).

Pattern G duplexes had the 2′F modification on pyrimidines of theantisense strand and 2′OMe modifications on pyrimidines in the sensestrand.

Pattern (H) had antisense strands with 2′F-modified pyrimidines in5′-PyPu-3′ dinucleotides, only at UA, CA, UG sites (both strands) andsense strands with 2′OMe modifications only on pyrimidines in 5′-PyPu-3′dinucleotides, only at UA, CA, UG sites (both strands).

A-D: Full Alternating 2′-OMe (both strands)

-   -   Four configurations: Even/Odd; Odd/Even; Even/Even; Odd/Odd

E: 2′-OMe Py (both strands)

F: 2′-OMe Py only at UA, CA, UG sites (both strands)

G: 2′-OMe All Py (sense)

-   -   2′-F All Py (anti-sense)

H: 2′-OMe Py only at UA, CA, UG sites (sense)

-   -   2′-F Py only at UA, CA, UG sites (anti-sense)

17 different parent VEGF duplexes from Table 2 tested

1. Evaluation of Serum Stability of siRNA Duplexes

2 μM siRNA duplexes (final concentration) were incubated in 90% pooledhuman serum at 37° C. Samples were quenched on dry ice after 30 minutes,4 hours, and 24 hours. For each siRNA sequence, a sample at the sameconcentration was incubated in the absence of serum (in PBS) at 37° C.for 24 hours. After all samples were quenched, RNA was extracted usingphenol:chloroform and concentrated by ethanol precipitation. Sampleswere air dried and resuspended in a denaturing loading buffer. One thirdof each time point was analyzed on a 20% acrylamide (19:1), 7 M urea,1×TBE gel run at 60° C. RNA was visualized by staining with stains-allsolution. A qualitative assessment of the stability of each modifiedsiRNA was made by comparison to the parent unmodified siRNA for eachduplex set. PBS controls served as markers for the quality of the inputsiRNA.

2. Stability of VEGF Modular Chemistries

Four modular chemistries were screened 1) all pyrimidines substitutedwith 2′-O-methyl (2′OMe) in both sense and antisense strands, 2)pyrimidines in UA, UG, CA pairs substituted with 2′OMe in both sense andantisense strands, 3) all pyrimidines substituted with 2′OMe in thesense strand and 2′-fluoro (2′F) in the antisense strand, 4) pyrimidinesin UA, UG, CA pairs substituted with 2′OMe in the sense strand and 2′Fin the antisense strand. In total, 85 siRNAs were screened including theunmodified parent duplexes plus the four modular chemistries.

Of the 85 siRNAs screened, 35 were stable for at least 24 hours asassessed by visual comparison with the parent unmodified duplexes. These35 duplexes had 2′OMe pyrimidines in both strands or 2′OMe pyrimidinesin the sense strand and 2′F in the antisense strand (chemistries 1 and 3above). Of the duplexes with fewer modified residues, only five had atleast ˜50% full length material remaining at the 4 hour time point ascompared to their unmodified parent.

Substitution of all pyrimidines with either 2′OMe or 2′F protects siRNAsfrom serum nuclease degradation for ˜24 hr in 90% human serum at 37° C.The protected duplexes had roughly 85%-100% full length materialremaining at 24 hours as compared to duplex incubated in the absence ofserum. Minimal modification of pyrimidines in UA, UG, and CAdinucleotide pairs only stabilized several siRNAs relative to theirunmodified parent but did not stabilize sufficiently for long-termnuclease resistance. Some potential RNase A sites were not protected bymethylation (YpN, e.g. UC, UU) and this is likely the reason for thelower resistance to serum endonucleases.

3. Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 6 and

FIGS. 13-29 provides summary and graphs of duplex activities in HeLacells for each of the modifications described above.

Synthesis of the iRNA Agents

RNA Synthesis Using “Fast” Deprotection Monomers

1. RNA Synthesis

Oligoribonucleotides were synthesized using phosphoramidite technologyon solid phase employing an AKTA 10 synthesizer (Amersham Biosciences)at scales ranging from 35 to 60 mmol. Synthesis was performed on solidsupports made of controlled pore glass (CPG, 520 {acute over (Å)}, witha loading of 70 μmol/g) or polystyrene (with a loading of 71 μmmol/g).All amidites were dissolved in anhydrous acetonitrile (70 mM) andmolecular sieves (3 {acute over (Å)}) were added. 5-Ethyl thiotetrazole(ETT, 600 mM in acetonitrile) was used as the activator solution.Coupling times were 8 minutes. Oxidation was carried out either with amixture of iodine/water/pyridine (50 mM/10%/90% (v/v)) or by employing a100 mM solution of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) inanhydrous acetonitrile in order to introduce phosphorothioate linkages.Standard capping reagents were used. Cholesterol was conjugated to RNAvia the either the 5′ or the 3′-end of the sense strand by starting froma CPG modified with cholesterol (described below) using ahydroxyprolinol linker. The DMT protecting group was removed fromcholesterol-conjugated RNA, but the DMT was left on unconjugated RNA tofacilitate purification.

2. Cleavage and Deprotection of Support Bound Oligonucleotide.

After solid-phase synthesis, the RNA was cleaved from the support bypassing 14 mL of a 3:1 (v/v) mixture of 40% methylamine in water andmethylamine in ethanol through the synthesis column over a 30 min timeperiod. For the cholesterol-conjugated RNA, the ratio of methylamine inwater to methylamine in ethanol was 1:13. The eluent was divided intofour 15 mL screw cap vials and heated to 65° C. for additional 30 min.This solution was subsequently dried down under reduced pressure in aspeedvac. The residue in each vial was dissolved in 250 μLN-methylpyrrolidin-2-one (NMP), and 120 μL triethylamine (TEA) and 160μL TEA.3HF were added. This mixture was brought to 65° C. for 2 h. Aftercooling to ambient temperature, 1.5 mL NMP and 1 mL ofethoxytrimethylsilane were added. After 10 min, the oligoribonucleotidewas precipitated by adding 3 mL of ether. The pellets were collected bycentrifugation, the supernatants were discarded, and the solids werereconstituted in 1 mL buffer 10 mM sodium phosphate.

3. Purification of Oligoribonucleotides

Crude oligonucleotides were purified by reversed phase HPLC on an AKTAExplorer system (Amersham Biosciences) using a 16/10 HR column (AmershamBiosciences) packed to a bed height of 10 cm with Source RPC 15. BufferA was 10 mM sodium phosphate and buffer B contained 65% acetonitrile inbuffer A. A flow rate of 6.5 mL/min was employed. UV traces at 260, 280,and 290 nm were recorded. For DMT-on oligoribonucleotides a gradient of7% B to 45% B within 10 column volumes (CV) was used and forcholesterol-conjugated RNA a gradient of 5% B to 100% B within 14 CV wasemployed. Appropriate fractions were pooled and concentrated underreduced pressure to roughly 10 mL. DMT-on oligonucleotides were treatedwith one-third volume 1M NaOAc, pH 4.25 for several hours at ambienttemp.

Finally, the purified oligonucleotides were desalted by size exclusionchromatography on a column containing Sephadex G-25. The oligonucleotidesolutions were concentrated to a volume <15 mL. The concentrations ofthe solutions were determined by measurement of the absorbance at 260 nmin a UV spectrophotometer. Until annealing the individual strands werestored as frozen solutions at −20° C.

4. Analysis of Oligoribonucleotides

Cholesterol conjugated RNA was analyzed by CGE and LC/MS. UnconjugatedRNA was also analyzed by IEX-HPLC. CGE analysis was performed on aBeckmanCoulter PACE MDQ CE instrument, equipped with a fixed wavelengthdetector at 254 nm. An eCap DNA capillary (BeckmanCoulter) with aneffective length of 20 cm was used. All single stranded RNA samples wereanalyzed under denaturing conditions containing 6 M urea (eCap ssDNA100Gel Buffer Kit, BeckmanCoulter) at 40° C. Samples were injectedelectrokinetically with 10 kV for 5-8 sec. The run voltage was 15 kV.

IEX HPLC analysis was performed on a Dionex BioLC system equipped with afixed wavelength detector (260 and 280 nm), column oven, autosampler,and internal degasser. A Dionex DNAPac P100 column (4*250 mm) was usedas at a flow rate of 1.0 mL/min and 30° C. Unconjugated RNA (20 μL, 1OD/mL concentration) was injected. Eluent A contained 20 mM Na₂HPO₄, 10mM NaBr, 10% acetonitrile, pH 11 and Eluent B was 1 M NaBr in Eluent A.The elution started with 20% B for 1 min and then a linear gradient witha target concentration of 80% B over 20 min was employed.

LC-MS analysis was performed on an Ettan μLC-system (AmershamBioscience) equipped with a Jetstream column heater and a fixedwavelength detector (254 nm). A ThermoFinnigan LCQ DecaXP ESI-MS systemwith micro-spray source and ion trap detector was coupled online to theHPLC. Oligonucleotide samples (25 μL sample, 1 OD/mL concentration inwater for unconjugated RNA and 40 μL for cholesterol-conjugated RNA)were injected onto a Waters Xterra C8 MS column (2.1×50 mm; 2.5 μmparticle size) with a flow rate of 200 μL/min at 60° C. Composition ofeluent A was 400 mM hexafluoroisopropanol (HFIP), 16.3 mM TEA in H₂O, pH7.9 and eluent B was methanol. For unconjugated RNA elution started with7% B for 3 min and then a gradient from 7% B to 25% B in 13 min wasused. For cholesterol-conjugated material the starting conditions were35% B for 3 min and then the concentration of eluent B was increased to75% B in 30 min. Analysis figures are provided in Table 6.

5. Annealing of Oligoribonucleotides

Complementary strands were annealed by combining equimolar RNAsolutions. The mixture was lyophilized and reconstituted with anappropriate volume of annealing buffer (100 mM NaCl, 20 mM sodiumphosphate, pH 6.8) to achieve the desired concentration. This solutionwas placed into a water bath at 95° C. and then cooled to ambient temp.within 3 h. Extent of duplex formation was monitored by native 10%polyacrylamide gel electrophoresis (PAGE) and bands were visualized bystaining with the “stains all” reagent (Sigma).

RNA Synthesis Using “Standard” Deprotection Monomers Including Ribo and2′-O-Methyl Phosphoramidites.

A. RNA/2′OMe (Thioate Ends)

The chimeric RNA molecules with 2′-OMe nucleotides were synthesized on a394 ABI machine using the standard cycle written by the manufacturerwith modifications to a few wait steps. The solid support was CPG (500A). The monomers were either RNA phosphoramidites or 2′ OMe RNAphosphoramidites with standard protecting groups and used atconcentrations of 0.15 M in acetonitrile (CH₃CN) unless otherwisestated. Specifically the RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite;the 2′OMe RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-O-methyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-methyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-methyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-O-methyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The coupling times were 10 min for all monomers. Details of the otherreagents are as follows: Activator: 5-(ethylthio)-1H-tetrazole (0.25M);Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10%N-methylimidazole/THF. Phosphate oxidation involved THBP (10% in ACN)for 10 min while phosphorothioate oxidation utilized 0.05 M EDITHreagent/acetonitrile. Detritylation was achieved with 3%TCA/dichloromethane. The DMT protecting group was removed after the laststep of the cycle.

After completion of synthesis the controlled pore glass (CPG) wastransferred to a screw cap, sterile microfuge tube. The oligonucleotidewas cleaved and simultaneously the base and phosphate groups deprotectedwith 1.0 mL of a mixture of ethanolic methylamine:ammonia (8 Mmethylamine in ethanol/30% aq ammonia) (1:1) for 5 hours at 55° C. Thetube was cooled briefly on ice and then the solution was transferred toa 5 mL centrifuge tube; this was followed by washing three times with0.25 mL of 50% acetonitrile. The tubes were cooled at −80° C. for 15min, before drying in a lyophilizer.

The white residue obtained was resuspended in 200 uL of NMP/Et₃N/Et₃N—HFand heated at 65° C. for 1.5 h to remove the TBDMS groups at the2′-position. The oligonucleotides were then precipitated in dry diethylether (400 uL) containing Et₃N (1%). The liquid was removed carefully toyield a pellet at the bottom of the tube. Residual ether was removed inthe speed vacuum to give the “crude” RNA as a white fluffy material.Samples were dissolved in 1 mL RNase free water and quantitated bymeasuring absorbance at 260 nm. This crude material was stored at −20°C.

The crude oligonucleotides were analyzed and purified by HPLC. The crudeoligonucleotides were analyzed and purified by Reverse Phase IonPair (RPIP) HPLC. The RP HPLC analysis was performed on a Gilson LC system,equipped with a fixed wavelength detector (260 and 280 nm), column oven,autosampler and internal degasser. An XTerra C18 column (4.6*250 mm) wasused at a flow rate of 1.0 mL/min at 65° C. RNA (20 μL for analyticalrun, 1 mL for a preparative run at 1 OD/mL concentration) was injected.Eluent A contained 0.1 M TEAAc, HPLC water, pH 7.0 and Eluent B was 0.1M TEAAc in HPLC water, 70% acetonitrile, pH 7.0. The elution startedwith 10% B for 2 min, followed by 25% B in 4 min and then a lineargradient with a target concentration of 50% B over another 30 min wasemployed. The purified dry oligonucleotides were then desalted usingSephadex G25M.

B. Synthesis of Oligonucleotides with 2′-Fluoro Modifications

The RNA molecules were synthesized on a 394 ABI machine using thestandard cycle written by the manufacturer with modifications to a fewwait steps. The solid support was CPG (500 A, TsT AG 001 from AMChemicals LLC and the rC and rU were from Prime Synthesis). The monomerswere either RNA phosphoramidites or 2′ F phosphoramidites with standardprotecting groups and used at concentrations of 0.15 M in acetonitrile(CH₃CN) unless otherwise stated. Specifically the RNA phosphoramiditeswere5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-β-tbutyldimethylsilyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite;the 2′F RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁴-acetyl-2′-fluoro-2′-deoxy-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-fluoro-2′-deoxy-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The coupling times were 10 min for all monomers. Details of the otherreagents are as follows: Activator: 5-ethyl thiotetrazole (0.25 M); CapA: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF;phosphate oxidation involved THBP (10% in ACN) for 10 min whilephosphorothioate oxidation utilized 0.05 M EDITH reagent/acetonitrile.Detritylation was achieved with 3% TCA/dichloromethane. The DMTprotecting group was removed after the last step of the cycle.

After completion of synthesis, CPG was transferred to a screw cap,sterile microfuge tube. The oligonucleotide was cleaved and the base andphosphate groups were simultaneously deprotected with 1.0 mL of amixture of ethanolic ammonia (1:3) for 7 hours at 55° C. The tube wascooled briefly on ice and then the solution was transferred to a 5 mLcentrifuge tube; this was followed by washing three times with 0.25 mLof 50% acetonitrile. The tubes were cooled at −80° C. for 15 min, beforedrying in a lyophilizer.

The white residue obtained was resuspended in 200 uL of NMP/Et₃N/Et₃N—HFand heated at 50° C. for 16 h to remove the TBDMS groups at the 2′position. The oligonucleotides were then precipitated in dry diethylether (400 uL) containing Et₃N (1%). The liquid was removed carefully toyield a pellet at the bottom of the tube. Residual ether was removed inthe speed vacuum to give the “crude” RNA as a white fluffy material.Samples were dissolved in 1 mL RNase free water and quantitated bymeasuring the absorbance at 260 nm. This crude material was stored at−20° C.

The crude oligonucleotides were analyzed and purified by HPLC. Thepurified dry oligonucleotides were then desalted using Sephadex G25M.

C. Synthesis of Phosphorothioate RNA Oligoribonucleotides

The oligonucleotides were synthesized on a 394 ABI machine (ALN 0208)using the standard 93 step cycle written by the manufacturer withmodifications to a few steps as described below. The solid support wascontrolled pore glass (CPG, 2 μmole rA CPG, 520 A, or rU CPG, 500 A).The monomers were RNA phosphoramidites with standard protecting groupsused at concentrations of 0.15 M in acetonitrile (CH₃CN) unlessotherwise stated. Specifically the RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The coupling times were 10 min. Details of the other reagents are asfollows: activator: 5-ethyl thiotetrazole (0.25M); Cap A: 5% aceticanhydride/THF/pyridine; Cap B:10% N-methylimidazole/THF; PS-oxidation,0.05M EDITH reagent/acetonitrile. Detritylation was achieved with 3%TCA/dichloromethane.

After completion of synthesis the CPG was transferred to a screw capsterile microfuge tube. The oligonucleotide was cleaved andsimultaneously the base and phosphate groups deprotected with 1.0 mL ofa mixture of ethanolic methylamine:ammonia (1:1) for 5 hours at 55° C.The tube was cooled briefly on ice and then the solution was transferredto a 5 mL centrifuge tube; this was followed by washing with 3×0.25 mLof 50% acetonitrile. The tubes were cooled at −80° C. for 15 min, beforedrying in a lyophilizer.

The white residue obtained was resuspended in 200 μL of TEA.3HF andheated at 65° C. for 1.5 h to remove the TBDMS groups at the2′-position. The oligonucleotides were then precipitated by addition of400 μL dry MeOH. The liquid was removed after spinning in amicrocentrifuge for 5 minutes on the highest speed available. Residualmethanol was removed in speed vacuum. Samples were dissolved in 1 mLRNase free water and quantitated by measuring the absorbance at 260 nm.The crude material was stored at −20° C. The oligonucleotides wereanalyzed and purified by HPLC and then desalted using Sephadex G25M.

Example 9 Synthesis of Oligonucleotides with Alternating 2′-F RNA and2′O-Me RNA (Table 7)

A. Synthesis of CPGs for 2′F.

CPGs of 5′-O-DMTr-2′-deoxy-2′-fluororibonucleosides with appropriatebase protection were synthesized as shown in Scheme A.5′-O-DMTr-2′-Deoxy-2′-fluoro-N^(tBz)-A and5′-β-DMTr-2′-Deoxy-2′-fluoro-N^(iBu)-G were synthesized as reported(Kawasaki et al., J. Med. Chem., 1993, 36, 831). Reaction of compounds1001 with succinic anhydride in the presence of DMAP inethylenedichloride yielded compound 1005. Compound 1005 was treated with2,2′-dithiobis(5-nitropyridine) (DTNP) and triphenylphosphine in thepresence of DMAP in acetonitrile-ethylenedichloride and subsequentlywith lcaa CPG as reported by Kumar et al. (Nucleosides & Nucleotides,1996, 15, 879) yielded the desired CPG 1009. Loading of the CPG wasdetermined as reported in the literature (Prakash et al., J. Org. Chem.,2002, 67, 357). CPGs of suitably protected 2′-deoxy-2′-fluoro A, C and Gwere obtained as described above (Scheme A).

The chimeric RNA molecules with alternating 2′-F RNA and 2′O-Me RNA weresynthesized on a 394 ABI machine using the standard cycle written by themanufacturer with modifications to a few wait steps. The solid supportwere CPG (500 A). The monomers were either 2′-F RNA phosphoramidites or2′ OMe RNA phosphoramidites with standard protecting groups and used atconcentrations of 0.15 M in acetonitrile (CH₃CN) unless otherwisestated. Specifically the 2′OMe RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-β-methyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-methyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-methyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-O-methyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The 2′F RNA phosphoramidites5′-O-Dimethoxytrityl-N⁴-acetyl-2′-fluoro-2′-deoxy-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-fluoro-2′-deoxy-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.5′-O-Dimethoxytrityl-2′-fluoro-N²-isobutyryl-2′-deoxy-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramiditeand5′-O-Dimethoxytrityl-2′-fluoro-N²-isobutyryl-2′-deoxy-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The coupling times were 10 min for all monomers. Details of the otherreagents are as follows: Activator: 5-ethyl thiotetrazole (0.25M); CapA: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF;phosphate oxidation involved 0.02M I₂/THF/H₂O, while PS-oxidation wascarried out using EDITH reagent as described above. Detritylation wasachieved with 3% TCA/dichloromethane. The final DMT protecting group wasremoved in the synthesizer.

After completion of synthesis the CPG was transferred to a screw cap,sterile microfuge tube. The oligonucleotide was cleaved and the base andphosphate groups were simultaneously deprotected with 1.0 mL of amixture of ethanolic:ammonia (1:3) for 7 hours at 55° C. The tube wascooled briefly on ice and then the solution was transferred to a 5 mLcentrifuge tube; this was followed by washing three times with 0.25 mLof 50% acetonitrile. The tubes were cooled at −80° C. for 15 min beforedrying in a lyophilizer to give the “crude” RNA as a white fluffymaterial. Samples were dissolved in 1 mL RNase free water andquantitated by measuring the absorbance at 260 nm. This crude materialwas stored at −20° C.

The crude oligonucleotides were analyzed and purified by 20%polyacrylimide denaturing gels. The purified dry oligonucleotides werethen desalted using Sephadex G25M (Amersham Biosciences).

B. Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 7 and FIG. 30 provides graphs of the activities in HeLacells for each of the modifications described above.

Example 10 Conjugated VEGF Molecules (Tables 8, 9, 10 and 18)

1. Synthesis:

The RNA molecules were synthesized on an ABI-394 machine (AppliedBiosystems) using the standard 93 step cycle written by the manufacturerwith modifications to a few wait steps as described below. The solidsupport was controlled pore glass (CPG, 1 umole, 500 A) and the monomerswere RNA phosphoramidites with standard protecting groups(5′-O-dimethoxytrityl-N6-benzoyl-2′-O-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-O-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite.All amidites were used at a concentration of 0.15M in acetonitrile(CH₃CN) and a coupling time of 6 min for unmodified and 2′-O-Me modifiedmonomers and 12 min for modified and conjugated monomers. 5-ethylthiotetrazole (0.25M) was used as an activator. For the PO-oxidationIodine/Water/Pyridine and for PS-oxidation Beaucage reagent (2%) inanhy. acetonitrile was used. The sulfurization time was about 6 min. Allsyntheses was performed on a 1 umole scale.

Wait or Coupling Reagents Concentration step Activator: 0.25M5-Ethylthio-1H-tetrazole 720 sec  PO-oxidation 0.02M Iodine inTHF/Water/ 20 sec Pyridine PO-oxidation 0.02M t-Butyl-hydrogen peroxide600 sec  PS-oxidation 2% Beaucage reagent/anhy. 360 Sec (200Acetonitrile sec, wait +30 sec pulse +130 sec wait Cap A 5% 5%Phenoxyacetic 20 sec anhydride/THF/pyridine Cap B 10%  10%N-methylimidazole/ 20 sec THF Detritylation 3% TCA Trichloro AceticAcid/ 70 sec dichloromethane

The following types of modifications were used to perform the synthesisusing these protocols:

1. Unmodified phosphodiester backbone (PO) only

2. Phosphorothioate (PS) only

3. 2′-O-Me, PS

4. 3′-Naproxen, 2′F-5Me-U, PS

5. 5′-Cholesterol, PS

6. 3′-Choletserol, PS

7. 2′F-5Me-U, PS

8. 3′-Biotin, 2′F-5Me-U, PS

9. 3′-cholanic acid, 2′F-5Me-U, PS

10. Methylphosphonate

11. C-5 allyamino rU

2. Deprotection-I (Nucleobase Deprotection)

After completion of the synthesis, the controlled pore glass (CPG) wastransferred to a screw cap vial or screw cap RNase free microfuge tube.The oligonucleotide was cleaved from the support and the base andphosphate protecting groups were simultaneously removed by using of amixture of ethanolic ammonia (ammonia (28-30%:ethanol (3:1))-(1.0 mL)for 15 h at 55° C. The vial was cooled briefly on ice and then theethanolic ammonia mixture was transferred to a new microfuge tube. TheCPG was washed with portions of deionized water (2×0.1 mL). Thesupernatant was combined, cooled in dry ice for 10 min and then dried ina speed vac.

3. Deprotection-II (Removal of 2′-O-TBDMS Group)

The white residue obtained was resuspended in a mixture oftriethylamine, triethylamine trihydrofluoride (TEA.3HF ca. 24% HF)) and1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65° C. for90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the2′-position. The reaction was then quenched withisopropoxytrimethylsilane (iPrOMe₃Si, 400 ul) and further incubated onthe heating block leaving the caps open for 10 min; This causes thevolatile isopropoxytrimethylsilylfluoride adduct to vaporize. Theresidual quenching reagent was removed by drying in a speed vac. 3%Triethylamine in diethyl ether (1.5 ml) was added. The mixture wassubjected to centrifugation. A pellet of RNA formed. The supernatant waspipetted out without disturbing the pellet. The pellet was dried in aspeed vac. The crude RNA was obtained as a white fluffy material in themicrofuge tube.

4. Quantitation of Crude Oligomer or Raw Analysis

Samples were dissolved in deionized water (1.0 mL) and quantitated asfollows: Blanking was first performed with water alone (1 mL). A sampleof the RNA solution (20 ul) was diluted with water (980 uL) and mixedwell in a microfuge tube, then transferred to a cuvette and theabsorbance reading was obtained at 260 nm. The crude material was drieddown and stored at −20° C.

5. MS Analysis:

The crude samples (0.1 OD) analyzed using LC-MS.

6. Purification of Oligomers

(a) Polyacrylamide Gel Electrophoresis (PAGE) Purification

The oligonucleotides were purified by vertical slab polyacrylamide gelelectrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth,N.H.). Electrophoresis grade acrylamide (40%),N,N′-methylene-bis(acrylamide) (BIS), ammonium persulfate (APS,N,N,N′N′-tetramethylenediamine (TEMED), bromophenol blue (BPB), xylenecyanol (XC) 10×TBE (0.89 M tris-hydroxy-methylaminomethane, borate pH8.3, 20 mM disodium ethylenediaminetetraacetate) were from NationalDiagnostics (Atlanta, Ga.). The 12% denaturing gel was prepared forpurification of unmodified and modified oligoribonucleotides. Thethickness of the preparative gels was 1.5 mm. Loading buffer was 80%formamide in 10×TBE. After removal of the glass plates, the gels werecovered with Saran Wrap® and placed over a fluorescent TLC plateilluminated by a hand-held UV lamp for visualization. The desired bandswere excised and shaken overnight in 2 mL of water or 0.03 M SodiumAcetate. The eluent was removed by drying in a speed vac.

(b) High Performance Liquid Chromatography (HPLC) Purification:

Condition A: Purification of unmodified, 2′-O-Me/PSOligoribonucleotides:

Amount of injected sample is about ˜100 OD.

Column: Dionex PA-100 Semiprep.

Buffer A: Water

Buffer B: 0.25 M Tris.Cl pH 8.0

Buffer C, 0.375 M Sod.Perchlorate

Heating: 65° C.

Time Flow Buffer A Buffer B Buffer C TotalYield Purity 0 5.00 88% 10%2.0% 40-60% 85-98% 3.0 5.00 88% 10% 2.0% 30.0 5.00 57.0 10% 33.0 35.05.00 88% 10% 2.0% 40.0 5.00 88% 10% 2.0%

Condition B: Protocols for Purification of 2′-O-Me/PSOligoribonucleotides:

Column: Dionex PA-100 Semiprep.

Buffer A: Water

Buffer B 0.25 M Tris.Cl pH 8.0

Buffer C 0.8 M Sod.Perchlorate

Heating: 65° C.

Total Time Flow Buffer A Buffer B Buffer C Yield Purity 0 5.00 88% 10%2.0% ~40-60% 85-98% 3.0 5.00 88% 10% 2.0% 30.0 5.00 57.0 10% 33.0 35.05.00 88% 10% 2.0% 40.0 5.00 88% 10% 2.0%

7. Desalting of Purified Oligomer

The purified dry oligomer was then desalted using Sephadex G-25 M. Thecartridge was conditioned with 10 mL of deionised water thrice. Finallythe purified oligomer dissolved thoroughly in 2.5 mL RNAse free waterwas applied to the cartridge with a very slow drop-wise elution. Thesalt free oligomer was eluted with 3.5 ml deionized water directly intoa screw cap vial. The purified RNA material was dried down in speed vacand stored at −20° C.

Biotin Conjugated siRNAs (Table 10)

1. Synthesis:

The RNA molecules were synthesized on an ABI-394 machine (AppliedBiosystems) using the standard 93 step cycle written by the manufacturerwith modifications to a few wait steps as described below. The solidsupport was controlled pore glass (CPG, 1 umole, 500 A) and the monomerswere RNA phosphoramidites with standard protecting groups(5′-O-dimethoxytritylN6-Benzoyl-2′O-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-O-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite.The modified CPG and amidites were synthesized using known methods andas described herein. All amidites were used at a concentration of 0.15Min acetonitrile (CH₃CN) and a coupling time of 6 min for unmodified and2′-O-Me monomers and 12 min for modified and conjugated monomers.5-Ethylthio-1H-tetrazole (0.25M) was used as an activator. For thePO-oxidation Iodine/Water/Pyridine and for PS-oxidation Beaucage reagent(2%) in anhy. acetonitrile was used. The sulfurization time is about 6min. For synthesis of 3′-biotin conjugated siRNAs, t-butyl-hydrogenperoxide was used as oxidizing agent (oxidation time 10 min).

Wait or Coupling Reagent Concentration step Activator: 0.25M5-Ethylthio-tetrazole 300 sec for unmodified and 720 sec for modifiedoligos. PO-oxidation 0.02M Iodine in THF/water/ 20 sec pyridinePO-oxidation 0.02M t-Butyl-hydrogen peroxide 600 sec  PS-oxidation 2%Beaucage reagent/anhy. 360 Sec (200 Acetonitrile sec, wait +30 sec pulse+130 sec wait Cap A 5% 5% Phenoxyacetic 20 sec anhydride/THF/pyridineCap B 10%  10% N- 20 sec Detritylation 3% TCA Methylimidazole/THF 70 secTrichloro Acetic Acid/dichloromethane

2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis the controlled pore glass (CPG) wastransferred to a screw cap vial or a screw cap RNase free microfugetube. The oligonucleotide was cleaved from the support with thesimultaneous removal of base and phosphate protecting groups with amixture of ethanolic ammonia [ammonia (28-30%):ethanol (3:1) 1.0 mL] for15 h at 55° C. The vial was cooled briefly on ice and then the ethanolicammonia mixture was transferred to a new microfuge tube. The CPG waswashed with portions of deionized water (2×0.1 mL). The combinedfiltrate was then put in dry ice for 10 min dried in a speed vac.

3. Deprotection-II (Removal of 2′-O-TBDMS Group)

The white residue obtained was resuspended in a mixture oftriethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) and1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65° C. for90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the2′-position. The reaction was then quenched withisopropoxytrimethylsilane (iPrOMe₃Si, 400 ul) and further incubated onthe heating block leaving the caps open for 10 min; (This causes thevolatile isopropxytrimethylsilylfluoride adduct to vaporize). Theresidual quenching reagent was removed by drying in a speed vac. 3%Triethylamine in diethyl ether (1.5 ml) was added and the mixture wassubjected to centrifugation to afford a pellet of RNA. The supernatantwas pipetted out without disturbing the pellet. The pellet was dried ina speed vac. The crude RNA was obtained as a white fluffy material inthe microfuge tube.

4. Quantitation of Crude Oligomer or Raw Analysis

Samples were dissolved in deionized water (1.0 mL) and quantitated asfollows: Blanking was first performed with water alone (1 mL). A sampleof the RNA solution (20 ul) was diluted with water (980 uL) and mixedwell in a microfuge tube, then transferred to a cuvette and theabsorbance reading was obtained at 260 nm. The crude material was drieddown and stored at −20° C.

5. MS Analysis:

Samples of the RNA (0.1 OD) were analyzed using MS.

6. Purification of Oligomers

Polyacrylamide Gel Electrophoresis (PAGE) Purification

The oligonucleotides were purified by vertical slab polyacrylamide gelelectrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth,N.H.). Electrophoresis grade acrylamide (40%),N,N′-methylene-bis(acrylamide) (BIS), ammonium persulfate (APS,N,N,N′N′-tetramethylenediamine (TEMED), bromophenol blue (BPB), xylenecyanol (XC) 10×TBE (0.89 M). Trishydroxy-methylaminomethane, borate (pH8.3), 20 mM disodium ethylenediaminetetraacetate) were from NationalDiagnostics (Atlanta, Ga.). The 12% Denaturing gel was prepared forpurification of oligoribonucleotides. The thickness of the preparativegel was 1.5 mm. Loading buffer was 80% formamide in 10×TBE. Afterremoval of the PAGE glass plates, the gels were covered with Saran Wrap®and placed over a fluorescent TLC plate illuminated by a hand-held UVlamp (Upland, Calif.) for visualization. The desired bands were excisedand shaken overnight in water (2 mL) or 0.03 M sodium acetate. Theeluent was removed and dried in a speed vac. All biotin conjugatedsequences were purified by PAGE.

7. Desalting of Purified Oligomer

The purified dry oligomer was then desalted using Sephadex G-25 M(Amersham Biosciences). The cartridge was conditioned with of deionizedwater thrice (10 mL each). Finally the purified oligomer dissolvedthoroughly in 2.5 mL RNAse free water was applied to the cartridge withvery slow drop-wise elution. The salt free oligomer was eluted withdeionized water (3.5 ml) directly into a screw cap vial. The purifiedRNA material was dried down on speed vac and stored at −20° C.

8. Quality Control

(a) Capillary Gel Electrophoresis (CGE)

(b) Electrospray LC/Ms

A sample of the oligomer (approx. 0.10 OD) was dissolved in water (50 ul& 100 ml in separate tubes) and then pipetted into special vials for CGEand LC/MS analysis.

9. Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Tables 8, 9, 10 and 18 and FIGS. 31-35 provides data and graphsof the activities in HeLa cells for each of the modifications describedabove.

Example 11 Conjugation of Retinoids to RNA (Table 11)

Conjugation of all-Trans-Retinal to Oligonucleotides (RNA):

Phoshoramidite 104 was synthesized as shown in Scheme B for retinalconjugation to oligonucleotides.

Step 1: Compound 102:

Monobenzylpentan-1,5-diol (15.70 g, 80.82 mmol), Ph₃P (25.43 g, 96.84mmol) and N-hydroxyphthalimide (116.0 g, 98.08 mmol) were taken inanhydrous CH₃CN (100 ml) under argon atm. Neat DIAD (20.0 mL, 103.25mmol) was added dropwise into the stirring solution over a period of 20minutes and the stirring was continued for 24 h. The reaction wasmonitored by TLC. Solvents were removed in vacuo; and the residue wastriturated with diethyl ether and filtered. Residue was washed withether, filtered and combined the filtrate. Hexane was added dropwiseinto the filtrate until it gave turbidity and subsequently the solutionwas made homogeneous by adding ether into it. The homogeneous solutionwas stored at 5° C. for 24 h. Precipitated Ph₃PO was filtered off,washed with ether-hexane mixture (1:1). Combined filtrate was evaporatedto dryness and the residue was purified by flash silica gel columnchromatography (10-15% EtOAc in Hexane) to obtain 24.5 g (89.3%) ofcompound 102 as a viscous pale yellow oil. ¹H NMR (400 MHz, CDCl₃, 25°C.): 7.84-7.82 (m, 2H); 7.75-7.73 (m, 2H); 7.34-7.33 (m, 4H); 7.29-7.26(m, 1H); 4.51 (s, 2H); 4.22-4.18 (t, J(H,H)=6.71 Hz, 2H); 3.52-3.48 (t,J(H,H)=6.4 Hz, 2H); 2.04-1.78 (m, 2H); 1.73-1.56 (m, 4H). ¹³C NMR (100MHz, CDCl₃, 25° C.): 163.9, 138.8, 134.6, 129.2, 128.6, 127.8, 127.7,123.7, 78.6, 73.1, 70.3, 29.6, 28.2, 22.5.

Step 2: Compound 103:

Compound 102 (23.5 g, 69.29 mmol) was taken in 100 ml of EtOAc/methanol(1:1). The mixture was degassed and purged with argon, to this 2.4 g ofPd—C (10%-wet Degusa type) was added. The mixture was then hydrogenatedovernight, filtered through a celite bed over a sintered funnel. Theresidue was subsequently passed through a column of silica gel andeluted out using 40% EtOAc in hexane to obtain compound 103 (15.70 g,90.9%) as a white solid. ¹H NMR (400 MHz, CDCl₃, 25° C.) 7.83-7.81 (bm.2H); 7.75-7.73 (bm, 2H); 4.23-4.19 (t, J(H,H)=6.4 Hz, 2H); 3.70-3.66 (t,J(H,H)=5.80 Hz, 2H); 1.83-1.79 (m, 2H); 1.67-1.60 (m, 4H). ¹³C NMR (100MHz, CDCl₃, 25° C.) 163.9, 134.7, 129.1, 123.7, 78.6, 62.7, 32.4, 28.0,22.0.

Step 3: Compound 104:

Compound 103 (5.4 g, 21.67 mmol) and triethylamine (4 ml, 28.69 mmol)were taken in anhydrous EtOAc (30 ml) under argon. 2-Cyanoethyldiisopropylchlorophosphoramidite (5.00 ml, 21.97 mmol) was added to thereaction mixture dropwise. A white precipitate of Et₃N.HCl was formedimmediately after the addition of the reagent and the reaction wascomplete in 10 min (monitored by TLC). The precipitate was filteredthrough a sintered funnel and solvent was removed under reducedpressure. The residue was directly loaded on a silica gel column forpurification. Eluted with hexane/EtOAc 9:1 to afford compound 104 as ayellow oil, 8.68 g (89.13%). ¹H NMR (400 MHz, CDCl₃, 25° C.) 7.85-7.81(m, 2H); δ 7.77-7.72 (m, 2H); 4.22-4.19 (t. J(H,H)=6.80 Hz, 2H);3.91-3.76 (m, 2H); 3.72-3.53 (m, 4H) 2.67-2.63 (t, J(H,H)=6.71 Hz, 2H);1.86-1.78 (m, 2H); 1.73-1.66 (m, 2H); 1.62-1.56 (m, 2H); 1.19-1.16 (m,12H). ³¹P NMR (162 MHz, CDCl₃, 25° C.) δ 145.09. ¹³C NMR (100 MHz,CDCl₃, 25° C.) δ 163.9, 134.7, 129.2, 123.7, 117.9, 78.6, 64.0, 63.4,58.7, 58.5, 43.2, 43.1, 31.1, 31.0, 28.1, 24.9, 24.8, 24.7, 22.3, 20.6,20.5.

Step 4: Conjugation of all-Trans-Retinal to Oligonucleotide:

All-trans-retinal was conjugated to oligonucleotide as shown in theScheme C. Compound 104 was coupled to solid bound oligonucleotide 105under standard solid phase oligonucleotide synthesis conditions toobtain compound 106. Phthalimido protecting group on compound 106 wasselectively removed by treating with hydrazinium hydrate as reported bySalo et al. (Bioconjugate Chem. 1999, 10, 815) to obtain compound 107.Treatment of compound 107 with all-trans-retinal under dark conditiongave compound 108 as reported in the literature (Bioconjugate Chem.1999, 10, 815). Standard RNA oligonucleotide deprotection andpurification under dark yielded the desired oligonucleotide-retinalconjugate 109. Compound 109 was also obtained from compound 110 as shownin Scheme C. Complete deprotection and purification of compound 106yielded an unbound free oligonucleotide 110 which was subsequentlyreacted with all-trans-retinal to afford the desired compound 109.

Step 4.1. Oligonucleotide Synthesis:

All oligonucleotides except AL-3166 were synthesized on an ABI 490 DNAsynthesizer. Commercially available controlled pore glass solid supports(dT-CPG and U-CPG, 500 {acute over (Å)}) and RNA phosphoramidites withstandard protecting groups,5′-O-dimethoxytrityl-N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditewere used for the oligonucleotide synthesis. All phosphoramidites wereused at a concentration of 0.15M in acetonitrile (CH₃CN). Coupling timeof 10 minutes was used. The activator was 5-ethyl thiotetrazole (0.25M),for the PO-oxidation Iodine/Water/Pyridine was used.

Sequence AL-3166 was synthesized on the AKTAoligopilot synthesizer. Allphosphoramidites were used at a concentration of 0.2M in acetonitrile(CH₃CN) except for guanosine which was used at 0.2M concentration in 10%THF/acetonitrile (v/v). Coupling/recycling time of 16 minutes was used.The activator was 5-ethyl thiotetrazole (0.75M), for the PO-oxidationIodine/Water/Pyridine was used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

The aminooxy-linker phosphoramidite was synthesized as described aboveand used at a concentration of 0.15M in acetonitrile. Coupling time forthe aminooxy-linker phosphoramidite was 15 minutes. For all sequences,coupling of the aminooxy-linker phosphoramidite was carried out on theABI 390 DNA synthesizer.

Step 4.2. Cleavage of the Phthalimido-Protecting Group from theAminoxy-Linker Oligonucleotides

After coupling of the aminooxy-linker, the CPG was treated with 2.5 mlof 0.5M hydrazinium acetate in pyridine (0.16/4/2 hydrazine anhydrous,pyridine, acetic acid) using the dual syringe method. Every 5 minutesthe syringes were pushed back and forth to get new solution on the CPG.After the hydrazinium acetate treatment, the CPG was washed with 2×5 mlof pyridine followed by 3×5 ml of acetonitrile. Flushing with dry argonfor 30 seconds then dried CPG.

Step 4.3. On Support Conjugation with the Aldehydes

The 1-pyrene-carboxaldehyde and the all-trans-retinal were from Aldrichand used at concentrations of 0.5M in DMF. The 4-keto-retinol was usedat a concentration of 0.13M in DMF. The CPG from above was added to thealdehyde solutions. Conjugation was carried out overnight (˜16 hrs) atroom temperature. After the reaction was complete, the CPG was rinsedwith DMF followed by acetonitrile and air dried for 10-15 minutes. Forsequence AL-3213, the conjugation with both all-trans-retinal and1-pyrene-carboxaldehyde was also carried out in acetonitrile. In thecase of 1-pyrene-carboxaldehyde, the aldehyde did not fully dissolved at0.5M and the solution was used as is without filtration to get rid ofthe undissolved aldehyde.

Step 4.4. Deprotection-I (Nucleobase Deprotection) of on SupportConjugated Oligonucleotides

For on support retinal conjugated oligonucleotides, the support wastransferred to a 5 ml tube (VWR). The oligonucleotide was cleaved fromthe support with simultaneous deprotection of base and phosphate groupswith 1 mL of 40% aq. methylamine 15 mins at 65° C. The tube was cooledbriefly on ice and then the methylamine was filtered into a new 15 mltube. The CPG was washed with 3×1 mL portions of DMSO.

Step 4.5. Deprotection-II (Removal of 2′ TBDMS Group) of on SupportConjugated Oligonucleotides

To the above mixture was added 1.5 ml of triethylamine trihydrofluoride(TREAT-HF) and heated at 60° C. for 15 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 5.5 ml of 50 mM sodium acetate (pH 5.5) andstored in freezer until purification.

Step 4.6. After Deprotection Conjugation with Aldehydes

Conjugation with the aldehydes (1-pyrene-carboxaldehyde andall-trans-retinal) after deprotection of the aminooxy-linkeroligonucleotides was also carried out as an alternative conjugationstrategy.

Step 4.7. Deprotection-I (Nucleobase Deprotection) for afterDeprotection Conjugation

The support was transferred to a 2 ml screw cap tube. Theoligonucleotide was cleaved from the support with simultaneousdeprotection of base and phosphate groups with 0.5 mL of 40% aq.methylamine 15 mins at 65° C. The tube was cooled briefly on ice andthen the methylamine was filtered into a new 15 ml tube. The CPG waswashed with 2×0.5 mL portions of 50% acetonitrile/water. The mixture wasthen frozen on dry ice and dried under vacuum on a speed vac.

Step 4.8. Deprotection-II (Removal of 2′ TBDMS Group) for afterDeprotection Conjugation

The dried residue was resuspended in 0.5 ml of triethylaminetrihydrofluoride (TEA.3HF) and heated at 60° C. for 15 minutes to removethe tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. Thereaction mixture was then cooled to room temperature and RNAprecipitated with 2 ml of dry methanol and dried under vacuum on a speedvac. The sample was then dissolved in 2 ml of water and kept frozen infreezer till further analysis.

Step 4.9. Quantitation of Crude Oligomer or Raw Analysis

For all samples, a 1 μl, a 10 μl or 30 μl aliquot was diluted with 999μl, 990 μl or 970 μl of deionised nuclease free water (1.0 mL) andabsorbance reading obtained at 260 nm.

Step 4.10. Purification of Conjugated Oligomers

(a) Cude LC/MS Analysis

The crude oligomers were first analyzed by LC/MS, to look at thepresence and abundance of the expected final product.

(b) Reverse-Phase Purification

The conjugated samples were purified by reverse-phase HPLC on anRPC-Source15 column (21.5×1 cm). The buffer system was: A=20 mM sodiumacetate in 10% ACN, pH 8.5 and B=20 mM sodium acetate in 70% ACN, pH8.5, with a flow rate of 5.0 mL/min, and wavelengths 260 and 375. Thefractions containing the full-length oligonucleotides were thenindividually desalted.

Step 4.11. Desalting of Purified Oligonucleotides

The purified oligonucleotide fractions were desalted using the PD-10Sephadex G-25 columns. First the columns were equilibrated with 25-30 mlof water. The samples were then applied in a volume of 2.5 ml. Thesamples were then eluted in salt-free fraction of 3.5 ml. The desaltedfractions were combined together and kept frozen till needed.

Step 4.12. Capillary Gel Electrophoresis (CGE), Ion-Exchange HPLC (IEX)and Electrospray LC/Ms

Approximately 0.3 OD of desalted oligonucleotides were diluted in waterto 300 μl and then pipetted in special vials for CGE, IEX and LC/MSanalysis.

Step 5 Conjugation of All-Trans-Retinal to 3′-End of Oligonucleotides(RNA):

Phoshoramidite 116 for 5′-conjugation and CPG support 115 for3′-conjugation of retinoids were synthesized as shown in the Scheme D.The CPG support 115 is used for 3′ conjugation of retinoids tooligonucleotides

Step 5.1: Compound 112: Compound III (120.0 g, 30.01 mmol) was stirredwith TBDMS-Cl (5.43 g, 36.02 mmol) in the presence of imidazole (7.5 g,110.16 mmol) in anhydrous pyridine (100 mL) overnight. After removingpyridine, the product was extracted into ethyl acetate (300 mL), washedwith aqueous sodium bicarbonate, followed by standard workup. Residueobtained was subjected to flash silica gel column chromatography using1% methanol in dichloromethane as eluent to afford compound 112 as apale white solid (24.4 g, qunat. ¹H NMR (500 MHz, [D₆]DMSO, 25° C.):7.33-7.13 (bm, 15H, accounted for 14H after D₂O exchange); 6.87-6.82(bm, 4H); 5.01 (s, 0.2H, rotamer minor); 4.99 (s, 1.8H, rotamer major),4.68-4.64 (m, 0.72 H, major rotamer); 4.14-4.07 (bm, 1H), 3.72 (s, 7H),3.38-3.36 (m, 0.6H, rotamer minor); 3.26-3.21 (m, 1.4H, rotamer major);3.08-3.07 (m, 0.3H, rotamer, minor); 2.99-2.89 (m, 2.7H, rotamer,major); 2.22-2.12 (m, 2H), 2.04-1.78 (m, 2H); 1.48-1.23 (m, 6H), 0.84,0.82 (s, 9H, rotamers major and minor); 0.05 (d, J(H,H)=1.5 Hz, 4.3H,rotamer major); 0.03-0.02 (d, J(H,H)=5.5 Hz, 1.7H).

Step 5.2: Compound 113: Compound 112 (9.4 g, 14.54 mmol) was suspendedin 15 mL of β-caprolactone and 10 mL of TEA was added into thesuspension. The reaction mixture was stirred under argon at 55° C. bathtemperature for 24 h. Completion of the reaction was monitored by TLCanalysis. TEA was removed form the reaction mixture in vacuo and 150 mLof dichloromethane-hexane (2:1 mixture) was added into the residue. Thehomogeneous solution thus obtained was directly loaded on a column ofsilica gel and eluted with dichloromethane-hexane (2:1) followed by neatdichloromethane. Elution of the silica column with 4% methanol indichloromethane afforded the desired compound 113 as a white solid (8.73g, 78.9%). ¹H NMR (400 MHz, [D₆]DMSO, 25° C.) δ 7.72-7.68 (bm, 1H,exchangeable with D₂O); 7.33-7.16 (m, 9H); 6.88-6.84 (m, 4H); 4.68-4.62(m, 0.8H); 4.57-4.52 (m, 0.2H); 4.34-4.31 (t, J(H,H)=5.18 Hz, 1H,exchangeable with D₂O); 4.14-4.08 (bm, 1H); 3.74-3.67 (m, 7H); 3.39-3.32(m, 3.3H); 3.25-3.21 (m, 1.7H); 3.09-2.88 (m, 4H)

6. Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 11 and FIG. 36 provide data and a graph of the activitiesin HeLa cells for each of the modifications described above.

Example 12 Conjugation of Polyethylene Glycol to siRNA (Table 12)

Amino Linker Oligonucleotides for PEG Conjugation

General.

Ion exchange preparative chromatography was performed onTSKgel-SuperQ-5PW (Tosoh). Ion exchange analytical chromatography wasperformed on a DNAPac Pa100 (Dionex). Electron spray ionization massspectra were recorded with an Agilent 1100 MSD-SL.

HPLC Techniques.

The RNA was analyzed by ion-exchange chromatography (column, DNAPacPa100, 4×250 mm, analytical), heated to 30° C., flow rate 1.5 mL min⁻¹,buffer A=0.020M Na₂HPO₄ in 10% CH₃CN, pH 11; buffer B=buffer A+1 M NaBrin 10% CH₃CN, pH 11, linear gradient from 0 to 75% in 53 min. TheLC/ESI-MS conditions were as follows: column XTerra C8 (2.1×30 mm, 2.5μm), linear gradient from 5 to 35% in 2 min and from 35 to 70% in 30.5min, flow rate 0.200 mL min⁻¹, buffer A=400 mM HFIP/16.3 mM TEA in H₂O,buffer B=100% methanol. The RNA was purified by ion-exchangechromatography (5 cm in-house packed column, TSKgel-SuperQ-5PW, 20 μm),heated to 75° C., flow rate 50 mL min⁻¹, buffer A=0.020M Na₂HPO₄ in 10%CH₃CN, pH 8.5; buffer B=buffer A+1 M NaBr in 10% CH₃CN, pH 8.5, lineargradient from 20 to 55% in 120 min.

RNA Synthesis.

The protected RNA was assembled on an AKTA Oligo Pilot 100 on a 100-150μmmol scale using custom in-house support and phosphoramidite chemistry.Phosphoramidites were used as 0.2 mol L⁻¹ solutions in dry CH₃CN, with a900 s coupling time and the manufacturer's recommended synthesisprotocols were used. After synthesis, the support-bound RNA was treatedwith aqueous CH₃NH₂ (40%) for 90 minutes at 45° C., cooled, filtered andwashed with DMSO (3×40 mL). The filtrate was then treated with TEA.3HF(60 mL) for 60 minutes at 40° C., and quenched with aq. NaOAc (0.05M, pH5.5, 200 mL). The synthesis was followed by analytical ion-exchangeHPLC, preparative HPLC, then desalting on Sephadex G-25.

Step 1. Oligonucleotide Synthesis:

A general conjugation approach is shown in the Scheme E.

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG,500{acute over (Å)}) or the phthalimido-hydroxy-prolinol solid supportand RNA phosphoramidites with standard protecting groups,5′-β-dimethoxytrityl-N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditewere used for the oligonucleotide synthesis. All phosphoramidites wereused at a concentration of 0.2M in acetonitrile (CH₃CN) except forguanosine which was used at 0.2M concentration in 10% THF/acetonitrile(v/v). Coupling/recycling time of 16 minutes was used. The activator was5-ethyl thiotetrazole (0.75M), for the PO-oxidationIodine/Water/Pyridine was used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used. The amino-linker phosphoramiditewas synthesized and used at a concentration of 0.2M in acetonitrile.Coupling/recycling time for the amino-linker phosphoramidite was 16minutes.

Step 2. Deprotection-I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle. The oligonucleotide was cleaved from the support withsimultaneous deprotection of base and phosphate groups with 40 mL of a40% aq. methyl amine 90 mins at 45° C. The bottle was cooled briefly onice and then the methylamine was filtered into a new 500 ml bottle. TheCPG was washed with 3×40 mL portions of DMSO. The mixture was thencooled on dry ice.

Step 3. Deprotection-II (Removal of 2′ TBDMS Group)

To the above mixture was added 60 ml triethylamine trihydrofluoride(TREAT-HF) and heated at 40° C. for 60 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 220 ml of 50 mM sodium acetate (pH 5.5) andstored in freezer until purification.

Step 4. Quantitation of Crude Oligomer or Raw Analysis

For all samples, a 10 μl aliquot was diluted with 990 μl of deionisednuclease free water (1.0 mL) and absorbance reading obtained at 260 nm.

Step 5. Purification of Oligomers

(a) HPLC Purification

The crude oligomers were first analyzed by HPLC (Dionex PA 100). Thebuffer system was: A=20 mM phosphate pH 11, B=20 mM phosphate, 1.8 MNaBr, pH 11, flow rate 1.0 mL/min, and wavelength 260-280 nm. Injectionsof 5-15 μl were done for each sample. The samples were purified by HPLCon an TSK-Gel SuperQ-5PW (20) column (17.3×5 cm). The buffer system was:A=20 mM phosphate in 10% ACN, pH 8.5 and B=20 mM phosphate, 1.0 M NaBrin 10% ACN, pH 8.5, with a flow rate of 50.0 mL/min, and wavelength 260and 294. The fractions containing the fulllength oligonucleotides werethen pooled together, evaporated and reconstituted to ˜100 ml withdeionised water.

Step 6. Desalting of Purified Oligomer

The purified oligonucleotides were desalted on an AKTA Explorer(Amersham Biosciences) using Sephadex G-25 column. First column waswashed with water at a flow rate of 25 ml/min for 20-30 min. The samplewas then applied in 25 ml fractions. The eluted salt-free fractions werecombined together, dried down and reconstituted in 50 ml of RNase freewater.

Step 7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms

Approximately 0.15 OD of desalted oligonucleotides were diluted in waterto 150 μl and then pipetted in special vials for CGE and LC/MS analysis.

Step 8. PEG Conjugation.

A) Initial Reaction Conditions.

The purified and desalted RNA was lyophilized. RNA (1 mg) was dissolvedin aq.NaHCO₃ (0.1M, 2004, pH 8.1) and DMF (2004 each). 5 K (13equivalents, 10 mg) or 20 KPEG (3.4 equivalents, 10 mg) was addeddirectly to reaction vial and vortexed thoroughly. The reactioncontinued overnight at 4° C., and was followed by analyticalion-exchange HPLC. When the reaction reached >85% completion, it wasquenched with aq. NaOAc (0.05M, pH 5.5) until the pH was ˜7.

B) Borate Buffer Conjugation.

The purified and desalted RNA was lyophilized. A sample of RNA (1 mg)was dissolved in sodium borate buffer (200 μL, 0.05M, pH10). 5 KPEG (3mg, 4.5 equivalents Sunbright ME-50HS, NOF Corp.) was dissolved in CH₃CN(200 μL). The RNA solution was added to the PEG solution and vortexedthoroughly. The reaction continued for one hour at room temperature, andwas followed by analytical ion-exchange HPLC. When reaction reached >85%completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pHwas ˜7.

C) PEG Linker (AS and HS) Comparison.

A sample of RNA (1 mg) was dissolved in aq, NaHCO₃ (0.1M, 2004, pH 8.1)and DMF (200 μL). 5 KPEG (13.5 eq, 10 mg, Sunbright ME-50HS or SunbrightME-50AS, NOF Corp.) was added directly to the reaction vial and vortexedthoroughly. The reaction continued overnight at 4° C., and was followedby analytical ion-exchange HPLC. When the reaction reached >85%completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pHwas ˜7.

D) Final Optimized PEG Conjugation.

The purified and desalted RNA was lyophilized. A sample of RNA (50 mg)was dissolved in aq. NaHCO₃ (0.1M, 2 mL pH 8.1) and DMF (1 mL). 20 KPEG(approximately 2.7 eq, 400-520 mg Sunbright ME-200HS, different amountsfor different sequences within this range) was dissolved in CH₃CN (2mL). The RNA solution was added to the PEG solution and vortexedthoroughly. H₂O (250 mL) was added to the reaction to decreaseturbidity. The reaction continued for one hour at room temperature, andwas followed by analytical ion-exchange HPLC. When the reactionreached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5)until the pH was ˜7.

Step 9. Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 12 provides data and graphs of the activities in HeLa cellsfor each of the modifications described above.

Example 13 Synthesis of Oligonucleotides Containing theRibo-Difluorotoluoyl (DFT) Nucleoside (Table 13)

The RNA molecules were synthesized on a 394 ABI machine using thestandard cycle written by the manufacturer with modifications to a fewwait steps. The solid support was 500 Å dT CPG (2 umole). The monomerswere either RNA phosphoramidites or the ribo-difluorotoluoyl amidite.All had standard protecting groups and were used at concentrations of0.15 M in acetonitrile (CH₃CN) unless otherwise stated. Specifically thephosphoramidites were5′-β-Dimethoxytrityl-N⁶-benzoyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-difluorotoluoylO-tbutyldimethylsilyl-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite(0.12 M). The coupling times were 7 min for all RNA monomers and 10 minfor the DFT monomer. Details of the other reagents are as follows:Activator: 5-ethylthio-1H-tetrazole (0.25M), Cap A: 5% aceticanhydride/THF/pyridine, Cap B: 10% N-methylimidazole/THF; phosphateoxidation involved 0.02M I₂/THF/H₂O. Detritylation was achieved with 3%TCA/dichloromethane. The DMT protecting group was removed after the laststep of the cycle.

After completion of synthesis the CPG was transferred to a screw cap,sterile microfuge tube. The oligonucleotide was cleaved and the base andphosphate groups were simultaneously deprotected with 1.0 mL of amixture of ethanolic ammonia (1:3) for 16 hours at 55° C. The tube wascooled briefly on ice and then the solution was transferred to a 5 mLcentrifuge tube; this was followed by washing three times with 0.25 mLof 50% acetonitrile. The tubes were cooled at −80° C. for 15 min, beforedrying in a lyophilizer.

The white residue obtained was resuspended in 200 uL of triethylaminetrihydrofluoride and heated at 65° C. for 1.5 h to remove the TBDMSgroups at the 2′-position. The oligonucleotides were then precipitatedin dry methanol (400 uL). The liquid was removed carefully to yield apellet at the bottom of the tube. Residual methanol was removed in thespeed vacuum to give a white fluffy material. Samples were dissolved in1 mL RNase free water and quantitated by measuring the absorbance at 260nm. This crude material was stored at −20° C.

The crude oligonucleotides were analyzed and purified by 20%polyacrylamide denaturing gels. The purified dry oligonucleotides werethen desalted using Sephadex G25M.

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 13 and FIG. 37 provide data and graphs of the activities inHeLa cells for each of the modifications described above.

Example 14 Synthesis of RNA Modified with2′-Ara-Fluoro-2′-Deoxy-Nucleosides (Table 14)

The chimeric RNA molecules were synthesized on a 394 ABI machine usingthe standard cycle written by the manufacturer with modifications to afew wait steps. The solid support was 500 Å dT CPG (2 μmole). Themonomers were either RNA phosphoramidites, or 2′-arafluoro-2′-deoxy (2′ara F) phosphoramidites. All monomers had standard protecting groups andwere used at concentrations of 0.15 M in acetonitrile (CH₃CN) unlessotherwise stated. Specifically the RNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-benzoyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(α-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-isobutyryl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite;the 2′ ara F phosphoramidites were5′-O-Dimethoxytrityl-N⁴-benzoyl-2′-arafluoro-2′-deoxy-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-arafluoro-2′-deoxy-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-arafluoro-thymidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite.The coupling times were 10 min for all monomers. Details of the otherreagents are as follows: Activator: 5-ethylthio-1H-tetrazole (0.25M),Cap A: 5% acetic anhydride/THF/pyridine, Cap B: 10%N-methylimidazole/THF; phosphate oxidation involved 0.02 M I₂/THF/H₂O.Detritylation was achieved with 3% TCA/dichloromethane. The final DMTprotecting group was removed after the last cycle.

After completion of synthesis the CPG was transferred to a screw cap,sterile microfuge tube. The oligonucleotide was cleaved and the base andphosphate groups were simultaneously deprotected with 1.0 mL of amixture of ethanolic ammonia conc (1:3) for 5 hours at 55° C. The tubewas cooled briefly on ice and then the solution was transferred to a 5mL centrifuge tube; this was followed by washing three times with 0.25mL of 50% acetonitrile. The tubes were cooled at −80° C. for 15 min,before drying in a lyophilizer.

The white residue obtained was resuspended in 200 μL of triethylaminetrihydrofluoride and heated at 65° C. for 1.5 h to remove the TBDMSgroups at the 2′-OH position. The oligonucleotides were thenprecipitated in dry methanol (400 μL). The liquid was removed carefullyto yield a pellet at the bottom of the tube. Residual methanol wasremoved in the speed vacuum to give a white fluffy material. Sampleswere dissolved in 1 mL RNase free water and quantitated by measuring theabsorbance at 260 nm. This crude material was stored at −20° C.

The crude oligonucleotides were analyzed and purified by 20%polyacrylamide denaturing gels. The purified dry oligonucleotides werethen desalted using Sephadex G25M (Amersham Biosciences).

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 14 and FIG. 38 provide data and graphs of the activities inHeLa cells for each of the modifications described above.

Example 15 Deprotection of Methylphosphonate Modified siRNAs (Table 15)

Deprotection Step 1:

After completion of the synthesis, the controlled pore glass (CPG) wastransferred to a screw cap vial. A solution (0.5 ml) consisting ofAcetonitrile/Ethanol/NH₄OH (45:45:10) was added to the support. The vialwas sealed and left at room temperature for 30 min. Ethylenediamine (0.5mL) was added to the vial and left at room temperature for an additional6 hours. The supernatant was decanted and the support was washed twicewith 1:1 acetonitrile/water (0.5 mL). The combined supernatant wasdiluted with water (15 mL). The pH was adjusted to 7.0 with 6 M HCl inAcCN/H₂O (1:9). The sample was desalted using a Sep-pak C₁₈ cartridgeand then dried in a speed vac.

Deprotection Step 2 (Removal of 2′-O-TBDMS Group)

The white residue obtained was resuspended in a mixture oftriethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) and1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65° C. for90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the2′-position. The reaction was then quenched withisopropoxytrimethylsilane (iPrOMe₃Si, 400 ul) and further incubated onthe heating block leaving the caps open for 10 min; (This causes thevolatile isopropxytrimethylsilylfluoride adduct to vaporize). Theresidual quenching reagent was removed by drying in a speed vac. 3%Triethylamine in diethyl ether (1.5 ml) was added and the mixture wassubjected to centrifugation to afford a pellet of RNA. The supernatantwas pipetted out without disturbing the pellet. The pellet was dried ina speed vac. The crude RNA was obtained as a white fluffy material inthe microfuge tube.

Purification:

All methylphosphonate modified sequences were purified by PAGE

Analysis of Duplex Activity

Duplexes were tested for activity in the HeLa cell assay describedabove. Table 15 provides data of the activities in HeLa cells for eachof the modifications described above.

Example 16 AL-DP-4409 Inhibits VEGF Protein and mRNA Expression in aDose-Dependent Manner

The ability of AL-DP-4409 to inhibit VEGF was assessed in vitro bytransient transfection of siRNAs into two human (HeLa and ARPE-19) celllines and one rat (RPE-J) cell line. AL-DP-4409 activity was compared toits unmodified “parent” siRNA (AL-DP-4094), its mismatch control(AL-DP-4409 MM), a control unrelated siRNA directed towards luciferase(AL-DP-3015), and an active VEGF siRNA reported in the literature(hVEFG5 or Candy (Reich et al. Molec. Vision 9:210-6, 2003)).

Methods: siRNA:

ALN-VEG01 is a chemically modified (one 3′ PS per strand) siRNA thattargets all VEGF-A spliced isoforms across multiple species (human, rat,mouse, etc.). ALN-VEG01 MM is a mismatch siRNA identical in sequence andchemistry to ALN-VEG01 with the exception of 5 mismatched base pairs.AL-DP-4094 represents the unmodified “parent” siRNA of ALN-VEG01 andcontains the identical sequence to ALN-VEG01 but with no chemicalmodifications. Control irrelevant siRNA targeting luciferase was alsoutilized. Sequences of the siRNAs are listed below (s indicatesphosphorothioate modification and mismatches are shown in bold):

AL-DP-4409: SEQ ID NO: 671 5′    GCACAUAGGAGAGAUGAGCUsU 3′SEQ ID NO: 939 3′ GsUCGUGUAUCCUCUCUACUCGA A 5′ AL-DP-4409 MM:SEQ ID NO: 1086 5′    GCACAU U GGA C AG U UG U G G UsU 3′SEQ ID NO: 1087 3′ GsUCGUGUA A CCU G UC A ACAC C A A 5′

Mismatches to AL-DP-4409 are shown as bold and underlined.

AL-DP-4094: SEQ ID NO: 608   5′ GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 6093′ GUCGUGUAUCCUCUCUACUCGAA 5′ AL-DP-3015: SEQ ID NO: 830   5′GAACUGUGUGUGAGAGGUCCU 3′ SEQ ID NO: 831 3′ CGCUUGACACACACUCUCCAGGA 5′

Transfection:

Human epithelial cervical adenocarcinoma (HeLa, ATCC, Cat#CCL-2.2) orretinal pigment epithelial cells—human (ARPE-19, ATCC, Manassas, Va.,Cat#CRL-2302) or rat (RPE-J, ATCC, Manassas, Va., Cat#CRL-2240), wereplated in 96-well plates (8,000-10,000 cells/well) in 100 μl 10% fetalbovine serum in Dulbecco's Modified Eagle Medium (DMEM, Gibco,Cat#11995-065). When the cells reached approximately 50% confluence (˜20hours later) they were transfected with serial three-fold dilutions ofsiRNA starting at 10 nM. 0.4 μl of transfection reagent Lipofectamine™2000 (Invitrogen Corporation, Carlsbad, Calif., Cat#11668-027) was usedper well and transfections were performed according to themanufacturer's protocol. Namely, the siRNA: Lipofectamine™ 2000complexes were prepared as follows. The appropriate amount of siRNA wasdiluted in Opti-MEM I Reduced Serum Medium (Gibco, Cat#31985-062). TheLipofectamine™ 2000 was mixed gently before use, then for each well of a96 well plate 0.4 μl was diluted in 25 μl of Opti-MEM I Reduced SerumMedium, mixed gently and incubated for 5 minutes at room temperature.After the 5 minute incubation, 1 μl of the diluted siRNA was combinedwith the diluted Lipofectamine™ 2000 (total volume is 26.4 μl). Thecomplex was mixed gently and incubated for 20 minutes at roomtemperature to allow the siRNA:Lipofectamine™ 2000 complexes to form.Then 100 μl of 10% fetal bovine serum in DMEM was added to each of thesiRNA:Lipofectamine™ 2000 complexes and mixed gently by rocking theplate back and forth. 100 μl of the above mixture was added to each wellcontaining the cells and the plates with human HeLa and ARPE-19 cellswere incubated at 37° C. (with rat RPE-J cells at −33° C.) in a CO₂incubator for 24 hours, then the culture medium was removed and 100 μlof fresh 10% fetal bovine serum in DMEM was added.

Following the medium change at time points indicated for eachexperiment, conditioned medium was collected to measure VEGF proteinlevels and cells were lysed to measure VEGF mRNA levels.

VEGF Protein Quantification in Conditioned Medium:

Human or rat VEGF ELISA was performed, using the Quantikine human or ratVEGF ELISA kit (R&D Systems, Inc. Minneapolis, Minn.). These kitscontain the basic components required for the development of sandwichELISAs to measure natural and recombinant human or rat VEGF in cellculture supernatants.

VEGF mRNA Quantification in Cell Lysates:

Cells were assayed for VEGF mRNA silencing using QuantiGene Discover Kit(Genospectra, Fremont, Calif., Cat#QG-000-010) following themanufacturer's protocol.

Results:

FIGS. 51 and 52 demonstrate that in human HeLa (2 dayspost-transfection) and ARPE-19 (5 days post-transfection) cell lines,respectively, both AL-DP-4409 and its originally synthesized batch(AL-DP-4094), but not Luciferase (AL-DP-3015) or AL-DP-4409 MM,inhibited VEGF protein expression in a dose-dependent manner. In bothcell lines, AL-DP-4409 exhibited more than 90% protein reduction with anIC50 of 100-200 nM, while VEGF5 (Cand5) exhibited less than 50% proteinreduction with an IC50 higher than 1 μM.

FIG. 53 demonstrates that in rat RPE-J cell line (7 dayspost-transfection) both AL-DP-4409 and its originally synthesizedunmodified batch (AL-DP-4094), but not Luciferase (AL-DP-3015) orAL-DP-4409 MM, inhibited VEGF protein expression in a dose-dependentmanner and exhibited more than 90% protein reduction with an IC50 of100-200 nM. VEGF5 (Cand5) does not crossreact with rat and did not showany activity.

On the mRNA level (FIG. 54), in HeLa cells, 2 days post-transfection,60% inhibition of VEGF mRNA was observed in a human HeLa cell line withAL-DP-4409 and its unmodified “parent” siRNA (AL-DP-4094), but not withLuciferase (AL-DP-3015) or AL-DP-4409 MM controls. The IC50 ofAL-DP-4409 was around 200 nM.

As shown in FIG. 55, VEGF protein inhibition by AL-DP-4409 was more than90% in human ARPE-19 cells, and this inhibition lasted for at least 10days when cells were treated with 30 nM AL-DP-4409. At doses less than0.4 nM, protein inhibition was maintained at more than 50% for at least10 days.

Example 17 An siRNA Directed Against VEGF Decreased RetinalNeovascularization

Rodent models of retinopathy of prematurity (ROP), also known asoxygen-induced retinopathy, have been utilized to assess the efficacy ofa variety of anti-angiogenic compounds in the context of ocularneovascularization.

Materials and Methods:

Newborn rats were exposed to oxygen concentration alternating between50% and 10% every 24 hrs from birth through day 14. Alternating oxygenconcentrations were used because the 50%/10% regimen produces a severe,reproducible, predictable and measurable retinal vascular response. Atday 14, the rats were removed to room air where they remained until day20. Therapeutic agents, including siRNA, were administeredintravitreally in PBS (5 μl/injection) at the time of removal from theexposure chamber (day 14). Following exposure to room air, the retinalvasculature responds to the “relative” low oxygen concentration of roomair as a hypoxic condition, thus resulting in the rapid upregulation ofangiogenic molecules, such as VEGF, which promote growth of new bloodvessels. Neovascularization was quantitatively assessed usingcomputer-assisted image analysis of flat mount retinal preparationsstained with ADPase. In addition, the area of retina containingintraretinal vessels was measured to determine the effect, if any, ofvarious treatments on the normal retinal vasculature. Neovascularizationdata are expressed as mean neovascular area (mm²)+/−SE and normalretinal vasculature data are expressed as % vascular area (+/−SE).Scheffe's post-hoc analysis was employed to identify significantdifferences in both neovascular area and normal vascular area.

Results. Dose-Dependent Response of AL-DP-4409 in Rat ROP Model.

Development of neovascularization was pronounced in rats exposed to roomair following alternating high oxygen exposure (FIG. 56).Neovascularization was pronounced in rats that were either untreated(n=6; mean=2.442±0.394 mm²) or injected intravitreally with PBS (n=5;mean=1.484±0.386 mm²) at day 14. Rats receiving an intravitrealinjection of a control siRNA directed against luciferase (AL-DP-3015)showed no effect of neovascularization as compared to control orbuffer-treated mice at any of the siRNA doses tested (60 μg; n=9;mean=1.449±0.320 mm²) (3 ug; n=3; mean=1.827±0.139 mm²) (0.15 μg; n=7;mean=2.249±0.233 mm²) (FIG. 56). In contrast, rats treated with a singleintravitreal injection of an siRNA directed against VEGF, identified asAL-DP-4409, showed a dramatic dose-dependent effect on retinalneovascularization (FIG. 56). Administration of 60 μg of AL-DP-4409 VEGFsiRNA decreased retinal neovascularization by 85% when compared withrats receiving 60 μg of control luciferase siRNA (n=7; mean=0.200±0.052mm²; p=0.001). Treatment with 3 μg of AL-DP-4409 VEGF siRNA was alsoeffective, reducing neovascularization by 65% when compared with ratsreceiving 3 μg of AL-DP-3015 control luciferase siRNA (n=3;mean=0.637±0.344 mm²); p=0.04). Treatment with 0.15 μg of AL-DP-4409VEGF siRNA had no effect on neovascularization (n=7; mean=1.790±0.127mm²), thus confirming the dose-responsive effect.

In contrast to the efficacy seen towards pathologic neovascularization,neither AL-DP-3015 control luciferase siRNA nor AL-DP-4409 VEGF siRNAtreatment had any effect on normal vascular development (FIG. 57).Confirmation of the selective and specific effect of AL-DP-4409 VEGFsiRNA on pathologic neovascularization and not on normal retinalvasculature was demonstrated in ADP-stained flat mount sections.

Specificity of AL-DP-4409 in Rat ROP Model.

To confirm the activity of AL-DP-4409 VEGF siRNA in inhibiting retinalneovascularization, AL-DP-4409 was compared with a specific mismatchedsiRNA, AL-DP-4409 MM (see above). The mismatch sequence results in lossof in vitro VEGF silencing activity. As demonstrated previously, robustdevelopment of retinal neovascularization was seen in animals thatreceived either no intravitreal injection (n=9; mean=1.996±0.151 mm²) orPBS buffer injection (n=7; mean=1.716±0.214 mm²) (FIG. 58). The VEGFmismatch siRNA showed no effect on neovascularization at a 60 μg dose(n=10; mean=2.012±0.200 mm²) (FIG. 58). Confirming previous results, the60 μg dose of AL-DP-4409 significantly inhibited neovascularization by75% (n=9; mean=0.503±0.086 mm²; p=0.001 vs MM), while the lower 3 μgdose of AL-Dp-4409 inhibited by 49% (n=9; mean=1.044±0.193 mm²; p=0.18vs MM). Lastly, none of the siRNA tested had any effect on normalretinal vasculature (FIG. 59).

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

TABLE 1 Target sequences in VEGF 121 TARGET SEQUENCE IN VEGF121 SEQ ID NO:  ORF mRNA 5′ to 3′ 2 1 AUGAACUUUCUGCUGUCUUGGGU 3 2UGAACUUUCUGCUGUCUUGGGUG 4 3 GAACUUUCUGCUGUCUUGGGUGC 5 4AACUUUCUGCUGUCUUGGGUGCA 6 5 ACUUUCUGCUGUCUUGGGUGCAU 7 6CUUUCUGCUGUCUUGGGUGCAUU 8 7 UUUCUGCUGUCUUGGGUGCAUUG 9 8UUCUGCUGUCUUGGGUGCAUUGG 10 9 UCUGCUGUCUUGGGUGCAUUGGA 11 10CUGCUGUCUUGGGUGCAUUGGAG 12 11 UGCUGUCUUGGGUGCAUUGGAGC 13 12GCUGUCUUGGGUGCAUUGGAGCC 14 13 CUGUCUUGGGUGCAUUGGAGCCU 15 14UGUCUUGGGUGCAUUGGAGCCUU 16 15 GUCUUGGGUGCAUUGGAGCCUUG 17 16UCUUGGGUGCAUUGGAGCCUUGC 18 17 CUUGGGUGCAUUGGAGCCUUGCC 19 18UUGGGUGCAUUGGAGCCUUGCCU 20 19 UGGGUGCAUUGGAGCCUUGCCUU 21 20GGGUGCAUUGGAGCCUUGCCUUG 22 21 GGUGCAUUGGAGCCUUGCCUUGC 23 22GUGCAUUGGAGCCUUGCCUUGCU 24 23 UGCAUUGGAGCCUUGCCUUGCUG 25 24GCAUUGGAGCCUUGCCUUGCUGC 26 25 CAUUGGAGCCUUGCCUUGCUGCU 27 26AUUGGAGCCUUGCCUUGCUGCUC 28 27 UUGGAGCCUUGCCUUGCUGCUCU 29 28UGGAGCCUUGCCUUGCUGCUCUA 30 29 GGAGCCUUGCCUUGCUGCUCUAC 31 30GAGCCUUGCCUUGCUGCUCUACC 32 31 AGCCUUGCCUUGCUGCUCUACCU 33 32GCCUUGCCUUGCUGCUCUACCUC 34 33 CCUUGCCUUGCUGCUCUACCUCC 35 34CUUGCCUUGCUGCUCUACCUCCA 36 35 UUGCCUUGCUGCUCUACCUCCAC 37 36UGCCUUGCUGCUCUACCUCCACC 38 37 GCCUUGCUGCUCUACCUCCACCA 39 38CCUUGCUGCUCUACCUCCACCAU 40 39 CUUGCUGCUCUACCUCCACCAUG 41 40UUGCUGCUCUACCUCCACCAUGC 42 41 UGCUGCUCUACCUCCACCAUGCC 43 42GCUGCUCUACCUCCACCAUGCCA 44 43 CUGCUCUACCUCCACCAUGCCAA 45 44UGCUCUACCUCCACCAUGCCAAG 46 45 GCUCUACCUCCACCAUGCCAAGU 47 46CUCUACCUCCACCAUGCCAAGUG 48 47 UCUACCUCCACCAUGCCAAGUGG 49 48CUACCUCCACCAUGCCAAGUGGU 50 49 UACCUCCACCAUGCCAAGUGGUC 51 50ACCUCCACCAUGCCAAGUGGUCC 52 51 CCUCCACCAUGCCAAGUGGUCCC 53 52CUCCACCAUGCCAAGUGGUCCCA 54 53 UCCACCAUGCCAAGUGGUCCCAG 55 54CCACCAUGCCAAGUGGUCCCAGG 56 55 CACCAUGCCAAGUGGUCCCAGGC 57 56ACCAUGCCAAGUGGUCCCAGGCU 58 57 CCAUGCCAAGUGGUCCCAGGCUG 59 58CAUGCCAAGUGGUCCCAGGCUGC 60 59 AUGCCAAGUGGUCCCAGGCUGCA 61 60UGCCAAGUGGUCCCAGGCUGCAC 62 61 GCCAAGUGGUCCCAGGCUGCACC 63 62CCAAGUGGUCCCAGGCUGCACCC 64 63 CAAGUGGUCCCAGGCUGCACCCA 65 64AAGUGGUCCCAGGCUGCACCCAU 66 65 AGUGGUCCCAGGCUGCACCCAUG 67 66GUGGUCCCAGGCUGCACCCAUGG 68 67 UGGUCCCAGGCUGCACCCAUGGC 69 68GGUCCCAGGCUGCACCCAUGGCA 70 69 GUCCCAGGCUGCACCCAUGGCAG 71 70UCCCAGGCUGCACCCAUGGCAGA 72 71 CCCAGGCUGCACCCAUGGCAGAA 73 72CCAGGCUGCACCCAUGGCAGAAG 74 73 CAGGCUGCACCCAUGGCAGAAGG 75 74AGGCUGCACCCAUGGCAGAAGGA 76 75 GGCUGCACCCAUGGCAGAAGGAG 77 76GCUGCACCCAUGGCAGAAGGAGG 78 77 CUGCACCCAUGGCAGAAGGAGGA 79 78UGCACCCAUGGCAGAAGGAGGAG 80 79 GCACCCAUGGCAGAAGGAGGAGG 81 80CACCCAUGGCAGAAGGAGGAGGG 82 81 ACCCAUGGCAGAAGGAGGAGGGC 83 82CCCAUGGCAGAAGGAGGAGGGCA 84 83 CCAUGGCAGAAGGAGGAGGGCAG 85 84CAUGGCAGAAGGAGGAGGGCAGA 86 85 AUGGCAGAAGGAGGAGGGCAGAA 87 86UGGCAGAAGGAGGAGGGCAGAAU 88 87 GGCAGAAGGAGGAGGGCAGAAUC 89 88GCAGAAGGAGGAGGGCAGAAUCA 90 89 CAGAAGGAGGAGGGCAGAAUCAU 91 90AGAAGGAGGAGGGCAGAAUCAUC 92 91 GAAGGAGGAGGGCAGAAUCAUCA 93 92AAGGAGGAGGGCAGAAUCAUCAC 94 93 AGGAGGAGGGCAGAAUCAUCACG 95 94GGAGGAGGGCAGAAUCAUCACGA 96 95 GAGGAGGGCAGAAUCAUCACGAA 97 96AGGAGGGCAGAAUCAUCACGAAG 98 97 GGAGGGCAGAAUCAUCACGAAGU 99 98GAGGGCAGAAUCAUCACGAAGUG 100 99 AGGGCAGAAUCAUCACGAAGUGG 101 100GGGCAGAAUCAUCACGAAGUGGU 102 101 GGCAGAAUCAUCACGAAGUGGUG 103 102GCAGAAUCAUCACGAAGUGGUGA 104 103 CAGAAUCAUCACGAAGUGGUGAA 105 104AGAAUCAUCACGAAGUGGUGAAG 106 105 GAAUCAUCACGAAGUGGUGAAGU 107 106AAUCAUCACGAAGUGGUGAAGUU 108 107 AUCAUCACGAAGUGGUGAAGUUC 109 108UCAUCACGAAGUGGUGAAGUUCA 110 109 CAUCACGAAGUGGUGAAGUUCAU 111 110AUCACGAAGUGGUGAAGUUCAUG 112 111 UCACGAAGUGGUGAAGUUCAUGG 113 112CACGAAGUGGUGAAGUUCAUGGA 114 113 ACGAAGUGGUGAAGUUCAUGGAU 115 114CGAAGUGGUGAAGUUCAUGGAUG 116 115 GAAGUGGUGAAGUUCAUGGAUGU 117 116AAGUGGUGAAGUUCAUGGAUGUC 118 117 AGUGGUGAAGUUCAUGGAUGUCU 119 118GUGGUGAAGUUCAUGGAUGUCUA 120 119 UGGUGAAGUUCAUGGAUGUCUAU 121 120GGUGAAGUUCAUGGAUGUCUAUC 122 121 GUGAAGUUCAUGGAUGUCUAUCA 123 122UGAAGUUCAUGGAUGUCUAUCAG 124 123 GAAGUUCAUGGAUGUCUAUCAGC 125 124AAGUUCAUGGAUGUCUAUCAGCG 126 125 AGUUCAUGGAUGUCUAUCAGCGC 127 126GUUCAUGGAUGUCUAUCAGCGCA 128 127 UUCAUGGAUGUCUAUCAGCGCAG 129 128UCAUGGAUGUCUAUCAGCGCAGC 130 129 CAUGGAUGUCUAUCAGCGCAGCU 131 130AUGGAUGUCUAUCAGCGCAGCUA 132 131 UGGAUGUCUAUCAGCGCAGCUAC 133 132GGAUGUCUAUCAGCGCAGCUACU 134 133 GAUGUCUAUCAGCGCAGCUACUG 135 134AUGUCUAUCAGCGCAGCUACUGC 136 135 UGUCUAUCAGCGCAGCUACUGCC 137 136GUCUAUCAGCGCAGCUACUGCCA 138 137 UCUAUCAGCGCAGCUACUGCCAU 139 138CUAUCAGCGCAGCUACUGCCAUC 140 139 UAUCAGCGCAGCUACUGCCAUCC 141 140AUCAGCGCAGCUACUGCCAUCCA 142 141 UCAGCGCAGCUACUGCCAUCCAA 143 142CAGCGCAGCUACUGCCAUCCAAU 144 143 AGCGCAGCUACUGCCAUCCAAUC 145 144GCGCAGCUACUGCCAUCCAAUCG 146 145 CGCAGCUACUGCCAUCCAAUCGA 147 146GCAGCUACUGCCAUCCAAUCGAG 148 147 CAGCUACUGCCAUCCAAUCGAGA 149 148AGCUACUGCCAUCCAAUCGAGAC 150 149 GCUACUGCCAUCCAAUCGAGACC 151 150CUACUGCCAUCCAAUCGAGACCC 152 151 UACUGCCAUCCAAUCGAGACCCU 153 152ACUGCCAUCCAAUCGAGACCCUG 154 153 CUGCCAUCCAAUCGAGACCCUGG 155 154UGCCAUCCAAUCGAGACCCUGGU 156 155 GCCAUCCAAUCGAGACCCUGGUG 157 156CCAUCCAAUCGAGACCCUGGUGG 158 157 CAUCCAAUCGAGACCCUGGUGGA 159 158AUCCAAUCGAGACCCUGGUGGAC 160 159 UCCAAUCGAGACCCUGGUGGACA 161 160CCAAUCGAGACCCUGGUGGACAU 162 161 CAAUCGAGACCCUGGUGGACAUC 163 162AAUCGAGACCCUGGUGGACAUCU 164 163 AUCGAGACCCUGGUGGACAUCUU 165 164UCGAGACCCUGGUGGACAUCUUC 166 165 CGAGACCCUGGUGGACAUCUUCC 167 166GAGACCCUGGUGGACAUCUUCCA 168 167 AGACCCUGGUGGACAUCUUCCAG 169 168GACCCUGGUGGACAUCUUCCAGG 170 169 ACCCUGGUGGACAUCUUCCAGGA 171 170CCCUGGUGGACAUCUUCCAGGAG 172 171 CCUGGUGGACAUCUUCCAGGAGU 173 172CUGGUGGACAUCUUCCAGGAGUA 174 173 UGGUGGACAUCUUCCAGGAGUAC 175 174GGUGGACAUCUUCCAGGAGUACC 176 175 GUGGACAUCUUCCAGGAGUACCC 177 176UGGACAUCUUCCAGGAGUACCCU 178 177 GGACAUCUUCCAGGAGUACCCUG 179 178GACAUCUUCCAGGAGUACCCUGA 180 179 ACAUCUUCCAGGAGUACCCUGAU 181 180CAUCUUCCAGGAGUACCCUGAUG 182 181 AUCUUCCAGGAGUACCCUGAUGA 183 182UCUUCCAGGAGUACCCUGAUGAG 184 183 CUUCCAGGAGUACCCUGAUGAGA 185 184UUCCAGGAGUACCCUGAUGAGAU 186 185 UCCAGGAGUACCCUGAUGAGAUC 187 186CCAGGAGUACCCUGAUGAGAUCG 188 187 CAGGAGUACCCUGAUGAGAUCGA 189 188AGGAGUACCCUGAUGAGAUCGAG 190 189 GGAGUACCCUGAUGAGAUCGAGU 191 190GAGUACCCUGAUGAGAUCGAGUA 192 191 AGUACCCUGAUGAGAUCGAGUAC 193 192GUACCCUGAUGAGAUCGAGUACA 194 193 UACCCUGAUGAGAUCGAGUACAU 195 194ACCCUGAUGAGAUCGAGUACAUC 196 195 CCCUGAUGAGAUCGAGUACAUCU 197 196CCUGAUGAGAUCGAGUACAUCUU 198 197 CUGAUGAGAUCGAGUACAUCUUC 199 198UGAUGAGAUCGAGUACAUCUUCA 200 199 GAUGAGAUCGAGUACAUCUUCAA 201 200AUGAGAUCGAGUACAUCUUCAAG 202 201 UGAGAUCGAGUACAUCUUCAAGC 203 202GAGAUCGAGUACAUCUUCAAGCC 204 203 AGAUCGAGUACAUCUUCAAGCCA 205 204GAUCGAGUACAUCUUCAAGCCAU 206 205 AUCGAGUACAUCUUCAAGCCAUC 207 206UCGAGUACAUCUUCAAGCCAUCC 208 207 CGAGUACAUCUUCAAGCCAUCCU 209 208GAGUACAUCUUCAAGCCAUCCUG 210 209 AGUACAUCUUCAAGCCAUCCUGU 211 210GUACAUCUUCAAGCCAUCCUGUG 212 211 UACAUCUUCAAGCCAUCCUGUGU 213 212ACAUCUUCAAGCCAUCCUGUGUG 214 213 CAUCUUCAAGCCAUCCUGUGUGC 215 214AUCUUCAAGCCAUCCUGUGUGCC 216 215 UCUUCAAGCCAUCCUGUGUGCCC 217 216CUUCAAGCCAUCCUGUGUGCCCC 218 217 UUCAAGCCAUCCUGUGUGCCCCU 219 218UCAAGCCAUCCUGUGUGCCCCUG 220 219 CAAGCCAUCCUGUGUGCCCCUGA 221 220AAGCCAUCCUGUGUGCCCCUGAU 222 221 AGCCAUCCUGUGUGCCCCUGAUG 223 222GCCAUCCUGUGUGCCCCUGAUGC 224 223 CCAUCCUGUGUGCCCCUGAUGCG 225 224CAUCCUGUGUGCCCCUGAUGCGA 226 225 AUCCUGUGUGCCCCUGAUGCGAU 227 226UCCUGUGUGCCCCUGAUGCGAUG 228 227 CCUGUGUGCCCCUGAUGCGAUGC 229 228CUGUGUGCCCCUGAUGCGAUGCG 230 229 UGUGUGCCCCUGAUGCGAUGCGG 231 230GUGUGCCCCUGAUGCGAUGCGGG 232 231 UGUGCCCCUGAUGCGAUGCGGGG 233 232GUGCCCCUGAUGCGAUGCGGGGG 234 233 UGCCCCUGAUGCGAUGCGGGGGC 235 234GCCCCUGAUGCGAUGCGGGGGCU 236 235 CCCCUGAUGCGAUGCGGGGGCUG 237 236CCCUGAUGCGAUGCGGGGGCUGC 238 237 CCUGAUGCGAUGCGGGGGCUGCU 239 238CUGAUGCGAUGCGGGGGCUGCUG 240 239 UGAUGCGAUGCGGGGGCUGCUGC 241 240GAUGCGAUGCGGGGGCUGCUGCA 242 241 AUGCGAUGCGGGGGCUGCUGCAA 243 242UGCGAUGCGGGGGCUGCUGCAAU 244 243 GCGAUGCGGGGGCUGCUGCAAUG 245 244CGAUGCGGGGGCUGCUGCAAUGA 246 245 GAUGCGGGGGCUGCUGCAAUGAC 247 246AUGCGGGGGCUGCUGCAAUGACG 248 247 UGCGGGGGCUGCUGCAAUGACGA 249 248GCGGGGGCUGCUGCAAUGACGAG 250 249 CGGGGGCUGCUGCAAUGACGAGG 251 250GGGGGCUGCUGCAAUGACGAGGG 252 251 GGGGCUGCUGCAAUGACGAGGGC 253 252GGGCUGCUGCAAUGACGAGGGCC 254 253 GGCUGCUGCAAUGACGAGGGCCU 255 254GCUGCUGCAAUGACGAGGGCCUG 256 255 CUGCUGCAAUGACGAGGGCCUGG 257 256UGCUGCAAUGACGAGGGCCUGGA 258 257 GCUGCAAUGACGAGGGCCUGGAG 259 258CUGCAAUGACGAGGGCCUGGAGU 260 259 UGCAAUGACGAGGGCCUGGAGUG 261 260GCAAUGACGAGGGCCUGGAGUGU 262 261 CAAUGACGAGGGCCUGGAGUGUG 263 262AAUGACGAGGGCCUGGAGUGUGU 264 263 AUGACGAGGGCCUGGAGUGUGUG 265 264UGACGAGGGCCUGGAGUGUGUGC 266 265 GACGAGGGCCUGGAGUGUGUGCC 267 266ACGAGGGCCUGGAGUGUGUGCCC 268 267 CGAGGGCCUGGAGUGUGUGCCCA 269 268GAGGGCCUGGAGUGUGUGCCCAC 270 269 AGGGCCUGGAGUGUGUGCCCACU 271 270GGGCCUGGAGUGUGUGCCCACUG 272 271 GGCCUGGAGUGUGUGCCCACUGA 273 272GCCUGGAGUGUGUGCCCACUGAG 274 273 CCUGGAGUGUGUGCCCACUGAGG 275 274CUGGAGUGUGUGCCCACUGAGGA 276 275 UGGAGUGUGUGCCCACUGAGGAG 277 276GGAGUGUGUGCCCACUGAGGAGU 278 277 GAGUGUGUGCCCACUGAGGAGUC 279 278AGUGUGUGCCCACUGAGGAGUCC 280 279 GUGUGUGCCCACUGAGGAGUCCA 281 280UGUGUGCCCACUGAGGAGUCCAA 282 281 GUGUGCCCACUGAGGAGUCCAAC 283 282UGUGCCCACUGAGGAGUCCAACA 284 283 GUGCCCACUGAGGAGUCCAACAU 285 284UGCCCACUGAGGAGUCCAACAUC 286 285 GCCCACUGAGGAGUCCAACAUCA 287 286CCCACUGAGGAGUCCAACAUCAC 288 287 CCACUGAGGAGUCCAACAUCACC 289 288CACUGAGGAGUCCAACAUCACCA 290 289 ACUGAGGAGUCCAACAUCACCAU 291 290CUGAGGAGUCCAACAUCACCAUG 292 291 UGAGGAGUCCAACAUCACCAUGC 293 292GAGGAGUCCAACAUCACCAUGCA 294 293 AGGAGUCCAACAUCACCAUGCAG 295 294GGAGUCCAACAUCACCAUGCAGA 296 295 GAGUCCAACAUCACCAUGCAGAU 297 296AGUCCAACAUCACCAUGCAGAUU 298 297 GUCCAACAUCACCAUGCAGAUUA 299 298UCCAACAUCACCAUGCAGAUUAU 300 299 CCAACAUCACCAUGCAGAUUAUG 301 300CAACAUCACCAUGCAGAUUAUGC 302 301 AACAUCACCAUGCAGAUUAUGCG 303 302ACAUCACCAUGCAGAUUAUGCGG 304 303 CAUCACCAUGCAGAUUAUGCGGA 305 304AUCACCAUGCAGAUUAUGCGGAU 306 305 UCACCAUGCAGAUUAUGCGGAUC 307 306CACCAUGCAGAUUAUGCGGAUCA 308 307 ACCAUGCAGAUUAUGCGGAUCAA 309 308CCAUGCAGAUUAUGCGGAUCAAA 310 309 CAUGCAGAUUAUGCGGAUCAAAC 311 310AUGCAGAUUAUGCGGAUCAAACC 312 311 UGCAGAUUAUGCGGAUCAAACCU 313 312GCAGAUUAUGCGGAUCAAACCUC 314 313 CAGAUUAUGCGGAUCAAACCUCA 315 314AGAUUAUGCGGAUCAAACCUCAC 316 315 GAUUAUGCGGAUCAAACCUCACC 317 316AUUAUGCGGAUCAAACCUCACCA 318 317 UUAUGCGGAUCAAACCUCACCAA 319 318UAUGCGGAUCAAACCUCACCAAG 320 319 AUGCGGAUCAAACCUCACCAAGG 321 320UGCGGAUCAAACCUCACCAAGGC 322 321 GCGGAUCAAACCUCACCAAGGCC 323 322CGGAUCAAACCUCACCAAGGCCA 324 323 GGAUCAAACCUCACCAAGGCCAG 325 324GAUCAAACCUCACCAAGGCCAGC 326 325 AUCAAACCUCACCAAGGCCAGCA 327 326UCAAACCUCACCAAGGCCAGCAC 328 327 CAAACCUCACCAAGGCCAGCACA 329 328AAACCUCACCAAGGCCAGCACAU 330 329 AACCUCACCAAGGCCAGCACAUA 331 330ACCUCACCAAGGCCAGCACAUAG 332 331 CCUCACCAAGGCCAGCACAUAGG 333 332CUCACCAAGGCCAGCACAUAGGA 334 333 UCACCAAGGCCAGCACAUAGGAG 335 334CACCAAGGCCAGCACAUAGGAGA 336 335 ACCAAGGCCAGCACAUAGGAGAG 337 336CCAAGGCCAGCACAUAGGAGAGA 338 337 CAAGGCCAGCACAUAGGAGAGAU 339 338AAGGCCAGCACAUAGGAGAGAUG 340 339 AGGCCAGCACAUAGGAGAGAUGA 341 340GGCCAGCACAUAGGAGAGAUGAG 342 341 GCCAGCACAUAGGAGAGAUGAGC 343 342CCAGCACAUAGGAGAGAUGAGCU 344 343 CAGCACAUAGGAGAGAUGAGCUU 345 344AGCACAUAGGAGAGAUGAGCUUC 346 345 GCACAUAGGAGAGAUGAGCUUCC 347 346CACAUAGGAGAGAUGAGCUUCCU 348 347 ACAUAGGAGAGAUGAGCUUCCUA 349 348CAUAGGAGAGAUGAGCUUCCUAC 350 349 AUAGGAGAGAUGAGCUUCCUACA 351 350UAGGAGAGAUGAGCUUCCUACAG 352 351 AGGAGAGAUGAGCUUCCUACAGC 353 352GGAGAGAUGAGCUUCCUACAGCA 354 353 GAGAGAUGAGCUUCCUACAGCAC 355 354AGAGAUGAGCUUCCUACAGCACA 356 355 GAGAUGAGCUUCCUACAGCACAA 357 356AGAUGAGCUUCCUACAGCACAAC 358 357 GAUGAGCUUCCUACAGCACAACA 359 358AUGAGCUUCCUACAGCACAACAA 360 359 UGAGCUUCCUACAGCACAACAAA 361 360GAGCUUCCUACAGCACAACAAAU 362 361 AGCUUCCUACAGCACAACAAAUG 363 362GCUUCCUACAGCACAACAAAUGU 364 363 CUUCCUACAGCACAACAAAUGUG 365 364UUCCUACAGCACAACAAAUGUGA 366 365 UCCUACAGCACAACAAAUGUGAA 367 366CCUACAGCACAACAAAUGUGAAU 368 367 CUACAGCACAACAAAUGUGAAUG 369 368UACAGCACAACAAAUGUGAAUGC 370 369 ACAGCACAACAAAUGUGAAUGCA 371 370CAGCACAACAAAUGUGAAUGCAG 372 371 AGCACAACAAAUGUGAAUGCAGA 373 372GCACAACAAAUGUGAAUGCAGAC 374 373 CACAACAAAUGUGAAUGCAGACC 375 374ACAACAAAUGUGAAUGCAGACCA 376 375 CAACAAAUGUGAAUGCAGACCAA 377 376AACAAAUGUGAAUGCAGACCAAA 378 377 ACAAAUGUGAAUGCAGACCAAAG 379 378CAAAUGUGAAUGCAGACCAAAGA 380 379 AAAUGUGAAUGCAGACCAAAGAA 381 380AAUGUGAAUGCAGACCAAAGAAA 382 381 AUGUGAAUGCAGACCAAAGAAAG 383 382UGUGAAUGCAGACCAAAGAAAGA 384 383 GUGAAUGCAGACCAAAGAAAGAU 385 384UGAAUGCAGACCAAAGAAAGAUA 386 385 GAAUGCAGACCAAAGAAAGAUAG 387 386AAUGCAGACCAAAGAAAGAUAGA 388 387 AUGCAGACCAAAGAAAGAUAGAG 389 388UGCAGACCAAAGAAAGAUAGAGC 390 389 GCAGACCAAAGAAAGAUAGAGCA 391 390CAGACCAAAGAAAGAUAGAGCAA 392 391 AGACCAAAGAAAGAUAGAGCAAG 393 392GACCAAAGAAAGAUAGAGCAAGA 394 393 ACCAAAGAAAGAUAGAGCAAGAC 395 394CCAAAGAAAGAUAGAGCAAGACA 396 395 CAAAGAAAGAUAGAGCAAGACAA 397 396AAAGAAAGAUAGAGCAAGACAAG 398 397 AAGAAAGAUAGAGCAAGACAAGA 399 398AGAAAGAUAGAGCAAGACAAGAA 400 399 GAAAGAUAGAGCAAGACAAGAAA 401 400AAAGAUAGAGCAAGACAAGAAAA

TABLE 2 Posi- SEQ SEQ EFFI- EFFI- tion ID Target sequence Alnylam IDCACY CACY in ORF NO: (5′-3′) DUP ID Strand NO: SEQUENCE HELA  HRPE 1 2AUGAACUUUCUGCUGUCUUGGGU AL-DP-4043 S 402 5 GAACUUUCUGCUGUCUUGGGU 3 +++NA AS 403 3 UACUUGAAAGACGACAGAACCCA 5 22 23 GUGCAUUGGAGCCUUGCCUUGCUAL-DP-4077 S 404 5 GCAUUGGAGCCUUGCCUUGCU 3 +++ NA AS 4053 CACGUAACCUCGGAACGGAACGA 5 47 48 UCUACCUCCACCAUGCCAAGUGG AL-DP-4021 S406 5 UACCUCCACCAUGCCAAGUTT 3 + NA AS 407 3 TTAUGGAGGUGGUACGGUUCA 5 4849 CUACCUCCACCAUGCCAAGUGGU AL-DP-4109 S 408 5 ACCUCCACCAUGCCAAGUGTT 3 +NA AS 409 3 TTUGGAGGUGGUACGGUUCAC 5 50 51 ACCUCCACCAUGCCAAGUGGUCCAL-DP-4006 S 410 5 CUCCACCAUGCCAAGUGGUCC 3 ++ + AS 4113 UGGAGGUGGUACGGUUCACCAGG 5 AL-DP-4083 S 412 5 CUCCACCAUGCCAAGUGGUTT 3++ ++ AS 413 3 TTGAGGUGGUACGGUUCACCA 5 51 52 CCUCCACCAUGCCAAGUGGUCCCAL-DP-4047 S 414 5 UCCACCAUGCCAAGUGGUCCC 3 + NA AS 4153 GGAGGUGGUACGGUUCACCAGGG 5 AL-DP-4017 S 416 5 UCCACCAUGCCAAGUGGUCTT 3 +NA AS 417 3 TTAGGUGGUACGGUUCACCAG 5 52 53 CUCCACCAUGCCAAGUGGUCCCAAL-DP-4048 S 418 5 CCACCAUGCCAAGUGGUCCCA 3 ++ ++ AS 4193 GAGGUGGUACGGUUCACCAGGGU 5 AL-DP-4103 S 420 5 CCACCAUGCCAAGUGGUCCTT 3++/+ ++ AS 421 3 TTGGUGGUACGGUUCACCAGG 5 53 54 UCCACCAUGCCAAGUGGUCCCAGAL-DP-4035 S 422 5 CACCAUGCCAAGUGGUCCCAG 3 ++ + AS 4233 AGGUGGUACGGUUCACCAGGGUC 5 AL-DP-4018 S 424 5 CACCAUGCCAAGUGGUCCCTT 3++/+ + AS 425 3 TTGUGGUACGGUUCACCAGGG 5 54 55 CCACCAUGCCAAGUGGUCCCAGGAL-DP-4036 S 426 5 ACCAUGCCAAGUGGUCCCAGG 3 +++ ++ AS 4273 GGUGGUACGGUUCACCAGGGUCC 5 AL-DP-4084 S 428 5 ACCAUGCCAAGUGGUCCCATT 3++ + AS 429 3 TTUGGUACGGUUCACCAGGGU 5 55 56 CACCAUGCCAAGUGGUCCCAGGCAL-DP-4093 S 430 5 CCAUGCCAAGUGGUCCCAGGC 3 ++ + AS 4313 GUGGUACGGUUCACCAGGGUCCG 5 AL-DP-4085 S 4325 CCAUGCCAAGUGGUCCCAGTT 3 + + AS 433 3 TTGGUACGGUUCACCAGGGUC 5 56 57ACCAUGCCAAGUGGUCCCAGGCU AL-DP-4037 S 434 5 CAUGCCAAGUGGUCCCAGGCU 3 + +AS 435 3 UGGUACGGUUCACCAGGGUCCGA 5 AL-DP-4054 S 4365 CAUGCCAAGUGGUCCCAGGTT 3 ++ + AS 437 3 TTGUACGGUUCACCAGGGUCC 5 57 58CCAUGCCAAGUGGUCCCAGGCUG AL-DP-4038 S 438 5 AUGCCAAGUGGUCCCAGGCUG 3 ++ ++AS 439 3 GGUACGGUUCACCAGGGUCCGAC 5 AL-DP-4086 S 4405 AUGCCAAGUGGUCCCAGGCTT 3 + + AS 441 3 TTUACGGUUCACCAGGGUCCG 5 58 59CAUGCCAAGUGGUCCCAGGCUGC AL-DP-4049 S 442 5 UGCCAAGUGGUCCCAGGCUGC 3 ++ ++AS 443 3 GUACGGUUCACCAGGGUCCGACG 5 AL-DP-4087 S 4445 UGCCAAGUGGUCCCAGGCUTT 3 + + AS 445 3 TTACGGUUCACCAGGGUCCGA 5 59 60AUGCCAAGUGGUCCCAGGCUGCA AL-DP-4001 S 446 5 GCCAAGUGGUCCCAGGCUGCA 3 ++ ++AS 447 3 UACGGUUCACCAGGGUCCGACGU 5 AL-DP-4052 A 4485 GCCAAGUGGUCCCAGGCUGTT 3 +++ ++ AS 449 3 TTCGGUUCACCAGGGUCCGAC 5 60 61UGCCAAGUGGUCCCAGGCUGCAC AL-DP-4007 S 450 5 CCAAGUGGUCCCAGGCUGCAC 3 +++++ AS 451 3 ACGGUUCACCAGGGUCCGACGUG 5 AL-DP-4088 S 4525 CCAAGUGGUCCCAGGCUGCTT 3 +++ ++ AS 453 3 TTGGUUCACCAGGGUCCGACG 5 61 62GCCAAGUGGUCCCAGGCUGCACC AL-DP-4070 S 454 5 CAAGUGGUCCCAGGCUGCACC 3 ++ ++AS 455 3 CGGUUCACCAGGGUCCGACGUGG 5 AL-DP-4055 S 4565 CAAGUGGUCCCAGGCUGCATT 3 +++ + AS 457 3 TTGUUCACCAGGGUCCGACGU 5 62 63CCAAGUGGUCCCAGGCUGCACCC AL-DP-4071 S 458 5 AAGUGGUCCCAGGCUGCACCC 3 + NAAS 459 3 GGUUCACCAGGGUCCGACGUGGG 5 AL-DP-4056 S 4605 AAGUGGUCCCAGGCUGCACTT 3 ++ NA AS 461 3 TTUUCACCAGGGUCCGACGUG 5 63 64CAAGUGGUCCCAGGCUGCACCCA AL-DP-4072 S 462 5 AGUGGUCCCAGGCUGCACCCA 3 ++ +AS 463 3 GUUCACCAGGGUCCGACGUGGGU 5 AL-DP-4057 S 4645 AGUGGUCCCAGGCUGCACCTT 3 ++/+ ++ AS 465 3 TTUCACCAGGGUCCGACGUGG 5 64 65AAGUGGUCCCAGGCUGCACCCAU AL-DP-4066 S 466 5 GUGGUCCCAGGCUGCACCCTT 3 + NAAS 467 3 TTCACCAGGGUCCGACGUGGG 5 99 100 AGGGCAGAAUCAUCACGAAGUGGAL-DP-4022 S 468 5 GGCAGAAUCAUCACGAAGUTT 3 +++ NA AS 4693 TTCCGUCUUAGUAGUGCUUCA 5 100 101 GGGCAGAAUCAUCACGAAGUGGU AL-DP-4023 S470 5 GCAGAAUCAUCACGAAGUGTT 3 ++ NA AS 471 3 TTCGUCUUAGUAGUGCUUCAC 5 101102 GGCAGAAUCAUCACGAAGUGGUG AL-DP-4024 S 472 5 CAGAAUCAUCACGAAGUGGTT 3 +NA AS 473 3 TTGUCUUAGUAGUGCUUCACC 5 102 103 GCAGAAUCAUCACGAAGUGGUGAAL-DP-4076 S 474 5 AGAAUCAUCACGAAGUGGUGA 3 ++ NA AS 4753 CGUCUUAGUAGUGCUUCACCACU 5 AL-DP-4019 S 476 5 AGAAUCAUCACGAAGUGGUTT 3++ NA AS 477 3 TTUCUUAGUAGUGCUUCACCA 5 103 104 CAGAAUCAUCACGAAGUGGUGAAAL-DP-4025 S 478 5 GAAUCAUCACGAAGUGGUGTT 3 ++ NA AS 4793 TTCUUAGUAGUGCUUCACCAC 5 104 105 AGAAUCAUCACGAAGUGGUGAAG AL-DP-4110 S480 5 AAUCAUCACGAAGUGGUGATT 3 + NA AS 481 3 TTUUAGUAGUGCUUCACCACU 5 105106 GAAUCAUCACGAAGUGGUGAAGU AL-DP-4068 S 482 5 AUCAUCACGAAGUGGUGAATT 3 +NA AS 483 3 TTUAGUAGUGCUUCACCACUU 5 113 114 ACGAAGUGGUGAAGUUCAUGGAUAL-DP-4078 S 484 5 GAAGUGGUGAAGUUCAUGGAU 3 +++ NA AS 4853 UGCUUCACCACUUCAAGUACCUA 5 121 122 GUGAAGUUCAUGGAUGUCUAUCA AL-DP-4080 S486 5 GAAGUUCAUGGAUGUCUAUCA 3 +++ NA AS 487 3 CACUUCAAGUACCUACAGAUAGU 5129 130 CAUGGAUGUCUAUCAGCGCAGCU AL-DP-4111 S 4885 UGGAUGUCUAUCAGCGCAGTT 3 +++ NA AS 489 3 TTACCUACAGAUAGUCGCGUC 5 130131 AUGGAUGUCUAUCAGCGCAGCUA AL-DP-4041 S 490 5 GGAUGUCUAUCAGCGCAGCUA 3+++ NA AS 491 3 UACCUACAGAUAGUCGCGUCGAU 5 AL-DP-4062 S 4925 GGAUGUCUAUCAGCGCAGCTT 3 +++ NA AS 493 3 TTCCUACAGAUAGUCGCGUCG 5 131132 UGGAUGUCUAUCAGCGCAGCUAC AL-DP-4069 S 494 5 GAUGUCUAUCAGCGCAGCUTT 3+++ NA AS 495 3 TTCUACAGAUAGUCGCGUCGA 5 132 133 GGAUGUCUAUCAGCGCAGCUACUAL-DP-4112 S 496 5 AUGUCUAUCAGCGCAGCUATT 3 + NA AS 4973 TTUACAGAUAGUCGCGUCGAU 5 133 134 GAUGUCUAUCAGCGCAGCUACUG AL-DP-4026 S498 5 UGUCUAUCAGCGCAGCUACTT 3 ++ NA AS 499 3 TTACAGAUAGUCGCGUCGAUG 5 134135 AUGUCUAUCAGCGCAGCUACUGC AL-DP-4095 S 500 5 GUCUAUCAGCGCAGCUACUGC 3+++ NA AS 501 3 UACAGAUAGUCGCGUCGAUGACG 5 AL-DP-4020 S 5025 GUCUAUCAGCGCAGCUACUTT 3 +++ NA AS 503 3 TTCAGAUAGUCGCGUCGAUGA 5 135136 UGUCUAUCAGCGCAGCUACUGCC AL-DP-4027 S 504 5 UCUAUCAGCGCAGCUACUGTT 3 +NA AS 505 3 TTAGAUAGUCGCGUCGAUGAC 5 144 145 GCGCAGCUACUGCCAUCCAAUCGAL-DP-4081 S 506 5 GCAGCUACUGCCAUCCAAUCG 3 +++ NA AS 5073 CGCGUCGAUGACGGUAGGUUAGC 5 146 147 GCAGCUACUGCCAUCCAAUCGAG AL-DP-4098 S508 5 AGCUACUGCCAUCCAAUCGAG 3 +++ NA AS 509 3 CGUCGAUGACGGUAGGUUAGCUC 5149 150 GCUACUGCCAUCCAAUCGAGACC AL-DP-4028 S 5105 UACUGCCAUCCAAUCGAGATT 3 ++ NA AS 511 3 TTAUGACGGUAGGUUAGCUCU 5 150 151CUACUGCCAUCCAAUCGAGACCC AL-DP-4029 S 512 5 ACUGCCAUCCAAUCGAGACTT 3 + NAAS 513 3 TTUGACGGUAGGUUAGCUCUG 5 151 152 UACUGCCAUCCAAUCGAGACCCUAL-DP-4030 S 514 5 CUGCCAUCCAAUCGAGACCTT 3 +++ NA AS 5153 TTGACGGUAGGUUAGCUCUGG 5 152 153 ACUGCCAUCCAAUCGAGACCCUG AL-DP-4031 S516 5 UGCCAUCCAAUCGAGACCCTT 3 + NA AS 517 3 TTACGGUAGGUUAGCUCUGGG 5 166167 GAGACCCUGGUGGACAUCUUCCA AL-DP-4008 S 518 5 GACCCUGGUGGACAUCUUCCA 3++ + AS 519 3 CUCUGGGACCACCUGUAGAAGGU 5 AL-DP-4058 S 5205 GACCCUGGUGGACAUCUUCTT 3 ++ ++ AS 521 3 TTCUGGGACCACCUGUAGAAG 5 167 168AGACCCUGGUGGACAUCUUCCAG AL-DP-4009 S 522 5 ACCCUGGUGGACAUCUUCCAG 3 ++ NAAS 523 3 UCUGGGACCACCUGUAGAAGGUC 5 AL-DP-4059 S 5245 ACCCUGGUGGACAUCUUCCTT 3 + NA AS 525 3 TTUGGGACCACCUGUAGAAGG 5 168 169GACCCUGGUGGACAUCUUCCAGG AL-DP-4010 S 526 5 CCCUGGUGGACAUCUUCCAGG 3 + +AS 527 3 CUGGGACCACCUGUAGAAGGUCC 5 AL-DP-4060 S 5285 CCCUGGUGGACAUCUUCCATT 3 +++ ++ AS 529 3 TTGGGACCACCUGUAGAAGGU 5 169170 ACCCUGGUGGACAUCUUCCAGGA AL-DP-4073 S 530 5 CCUGGUGGACAUCUUCCAGGA 3++ + AS 531 3 UGGGACCACCUGUAGAAGGUCCU 5 AL-DP-4104 S 5325 CCUGGUGGACAUCUUCCAGTT 3 +++/+ ++ AS 533 3 TTGGACCACCUGUAGAAGGUC 5 170171 CCCUGGUGGACAUCUUCCAGGAG AL-DP-4011 S 534 5 CUGGUGGACAUCUUCCAGGAG 3 +NA AS 535 3 GGGACCACCUGUAGAAGGUCCUC 5 AL-DP-4089 S 5365 CUGGUGGACAUCUUCCAGGTT 3 + NA AS 537 3 TTGACCACCUGUAGAAGGUCC 5 171 172CCUGGUGGACAUCUUCCAGGAGU AL-DP-4074 S 538 5 UGGUGGACAUCUUCCAGGAGU 3 ++ +AS 539 3 GGACCACCUGUAGAAGGUCCUCA 5 AL-DP-4090 S 5405 UGGUGGACAUCUUCCAGGATT 3 ++ ++ AS 541 3 TTACCACCUGUAGAAGGUCCU 5 172 173CUGGUGGACAUCUUCCAGGAGUA AL-DP-4039 S 542 5 GGUGGACAUCUUCCAGGAGUA 3 ++ ++AS 543 3 GACCACCUGUAGAAGGUCCUCAU 5 AL-DP-4091 S 5445 GGUGGACAUCUUCCAGGAGTT 3 + + AS 545 3 TTCCACCUGUAGAAGGUCCUC 5 175 176GUGGACAUCUUCCAGGAGUACCC AL-DP-4003 S 546 5 GGACAUCUUCCAGGAGUACCC 3 ++ ++AS 547 3 CCUGUAGAAGGUCCUCAUGGG 5 AL-DP-4116 S 5485 GGACAUCUUCCAGGAGUACCC 3 + NA AS 549 3 CCUGUAGAAGGUCCUCAUGGG 5AL-DP-4015 S 550 5 GGACAUCUUCCAGGAGUACTT 3 ++ ++ AS 5513 TTCCUGUAGAAGGUCCUCAUG 5 AL-DP-4120 S 552 5 GGACAUCUUCCAGGAGUAC 3 + NAAS 553 3 CCUGUAGAAGGUCCUCAUG 5 179 180 ACAUCUUCCAGGAGUACCCUGAUAL-DP-4099 S 554 5 AUCUUCCAGGAGUACCCUGAU 3 +++ NA AS 5553 UGUAGAAGGUCCUCAUGGGACUA 5 191 192 AGUACCCUGAUGAGAUCGAGUAC AL-DP-4032 S556 5 UACCCUGAUGAGAUCGAGUTT 3 +++ NA AS 557 3 TTAUGGGACUACUCUAGCUCA 5192 193 GUACCCUGAUGAGAUCGAGUACA AL-DP-4042 S 5585 ACCCUGAUGAGAUCGAGUACA 3 +++ NA AS 559 3 CAUGGGACUACUCUAGCUCAUGU 5AL-DP-4063 S 560 5 ACCCUGAUGAGAUCGAGUATT 3 +++ NA AS 5613 TTUGGGACUACUCUAGCUCAU 5 209 210 AGUACAUCUUCAAGCCAUCCUGU AL-DP-4064 S562 5 UACAUCUUCAAGCCAUCCUTT 3 + NA AS 563 3 TTAUGUAGAAGUUCGGUAGGA 5 260261 GCAAUGACGAGGGCCUGGAGUGU AL-DP-4044 S 564 5 AAUGACGAGGGCCUGGAGUGU 3 +NA AS 565 3 CGUUACUGCUCCCGGACCUCACA 5 263 264 AUGACGAGGGCCUGGAGUGUGUGAL-DP-4045 S 566 5 GACGAGGGCCUGGAGUGUGUG 3 + NA AS 5673 UACUGCUCCCGGACCUCACACAC 5 279 280 GUGUGUGCCCACUGAGGAGUCCA AL-DP-4046 S568 5 GUGUGCCCACUGAGGAGUCCA 3 +++ NA AS 569 3 CACACACGGGUGACUCCUCAGGU 5281 282 GUGUGCCCACUGAGGAGUCCAAC AL-DP-4096 S 5705 GUGCCCACUGAGGAGUCCAAC 3 +++ NA AS 571 3 CACACGGGUGACUCCUCAGGUUG 5 283284 GUGCCCACUGAGGAGUCCAACAU AL-DP-4040 S 572 5 GCCCACUGAGGAGUCCAACAU 3+++ NA AS 573 3 CACGGGUGACUCCUCAGGUUGUA 5 289 290ACUGAGGAGUCCAACAUCACCAU AL-DP-4065 S 574 5 UGAGGAGUCCAACAUCACCTT 3 + NAAS 575 3 TTACUCCUCAGGUUGUAGUGG 5 302 303 ACAUCACCAUGCAGAUUAUGCGGAL-DP-4100 S 576 5 AUCACCAUGCAGAUUAUGCGG 3 ++ NA AS 5773 UGUAGUGGUACGUCUAAUACGCC 5 305 306 UCACCAUGCAGAUUAUGCGGAUC AL-DP-4033 S578 5 ACCAUGCAGAUUAUGCGGATT 3 ++ NA AS 579 3 TTUGGUACGUCUAAUACGCCU 5 310311 AUGCAGAUUAUGCGGAUCAAACC AL-DP-4101 S 580 5 GCAGAUUAUGCGGAUCAAACC 3+++ NA AS 581 3 UACGUCUAAUACGCCUAGUUUGG 5 312 313GCAGAUUAUGCGGAUCAAACCUC AL-DP-4102 S 582 5 AGAUUAUGCGGAUCAAACCUC 3 +++NA AS 583 3 CGUCUAAUACGCCUAGUUUGGAG 5 315 316 GAUUAUGCGGAUCAAACCUCACCAL-DP-4034 S 584 5 UUAUGCGGAUCAAACCUCATT 3 ++ NA AS 5853 TTAAUACGCCUAGUUUGGAGU 5 316 317 AUUAUGCGGAUCAAACCUCACCA AL-DP-4113 S586 5 UAUGCGGAUCAAACCUCACTT 3 ++ NA AS 587 3 TTAUACGCCUAGUUUGGAGUG 5 317318 UUAUGCGGAUCAAACCUCACCAA AL-DP-4114 S 588 5 AUGCGGAUCAAACCUCACCTT 3 +NA AS 589 3 TTUACGCCUAGUUUGGAGUGG 5 319 320 AUGCGGAUCAAACCUCACCAAGGAL-DP-4002 S 590 5 GCGGAUCAAACCUCACCAAGG 3 +++ +++ AS 5913 UACGCCUAGUUUGGAGUGGUUCC 5 AL-DP-4115 S 592 5 GCGGAUCAAACCUCACCAA 3 +++NA AS 593 3 CGCCUAGUUUGGAGUGGUU 5 AL-DP-4014 S 5945 GCGGAUCAAACCUCACCAATT 3 +++ +++ AS 595 3 TTCGCCUAGUUUGGAGUGGUU 5AL-DP-4119 S 596 5 GCGGAUCAAACCUCACCAA 3 +++ NA AS 5973 CGCCUAGUUUGGAGUGGUU 5 321 322 GCGGAUCAAACCUCACCAAGGCC AL-DP-4013 S 5985 GGAUCAAACCUCACCAAGGCC 3 ++ NA AS 599 3 CGCCUAGUUUGGAGUGGUUCCGG 5 341342 GCCAGCACAUAGGAGAGAUGAGC AL-DP-4075 S 600 5 CAGCACAUAGGAGAGAUGAGC 3+++ ++ AS 601 3 CGGUCGUGUAUCCUCUCUACUCG 5 AL-DP-4105 S 6025 CAGCACAUAGGAGAGAUGATT 3 ++ ++ AS 603 3 TTGUCGUGUAUCCUCUCUACU 5 342 343CCAGCACAUAGGAGAGAUGAGCU AL-DP-4050 S 604 5 AGCACAUAGGAGAGAUGAGCU 3 ++++++ AS 605 3 GGUCGUGUAUCCUCUCUACUCGA 5 AL-DP-4106 S 6065 AGCACAUAGGAGAGAUGAGTT 3 ++ +++ AS 607 3 TTUCGUGUAUCCUCUCUACUC 5 343344 CAGCACAUAGGAGAGAUGAGCUU AL-DP-4094 S 608 5 GCACAUAGGAGAGAUGAGCUU 3+++ +++ AS 609 3 GUCGUGUAUCCUCUCUACUCGAA 5 AL-DP-4118 S 6105 GCACAUAGGAGAGAUGAGCUU 3 * NA AS 611 3 CGUGUAUCCUCUCUACUCGAA 5AL-DP-4107 S 612 5 GCACAUAGGAGAGAUGAGCTT 3 +++ +++ AS 6133 TTCGUGUAUCCUCUCUACUCG 5 AL-DP-4122 S 614 5 GCACAUAGGAGAGAUGAGC 3 ++ NAAS 615 3 CGUGUAUCCUCUCUACUCG 5 344 345 AGCACAUAGGAGAGAUGAGCUUCAL-DP-4012 S 616 5 CACAUAGGAGAGAUGAGCUUC 3 +++ +++ AS 6173 UCGUGUAUCCUCUCUACUCGAAG 5 AL-DP-4108 S 618 5 CACAUAGGAGAGAUGAGCUTT 3+++ +++ AS 619 3 TTGUGUAUCCUCUCUACUCGA 5 346 347 CACAUAGGAGAGAUGAGCUUCCUAL-DP-4051 S 620 5 CAUAGGAGAGAUGAGCUUCCU 3 +++ +++ AS 6213 GUGUAUCCUCUCUACUCGAAGGA 5 AL-DP-4061 S 622 5 CAUAGGAGAGAUGAGCUUCTT 3+++ +++ AS 623 3 TTGUAUCCUCUCUACUCGAAG 5 349 350 AUAGGAGAGAUGAGCUUCCUACAAL-DP-4082 S 624 5 AGGAGAGAUGAGCUUCCUACA 3 +++ NA AS 6253 UAUCCUCUCUACUCGAAGGAUGU 5 369 370 ACAGCACAACAAAUGUGAAUGCA AL-DP-4079 S626 5 AGCACAACAAAUGUGAAUGCA 3 ++ NA AS 627 3 UGUCGUGUUGUUUACACUUACGU 5372 373 GCACAACAAAUGUGAAUGCAGAC AL-DP-4097 S 6285 ACAACAAAUGUGAAUGCAGAC 3 ++ NA AS 629 3 CGUGUUGUUUACACUUACGUCUG 5 379380 AAAUGUGAAUGCAGACCAAAGAA AL-DP-4067 S 630 5 AUGUGAAUGCAGACCAAAGTT 3++ NA AS 631 3 TTUACACUUACGUCUGGUUUC 5 380 381 AAUGUGAAUGCAGACCAAAGAAAAL-DP-4092 S 632 5 UGUGAAUGCAGACCAAAGATT 3 +++ NA AS 6333 TTACACUUACGUCUGGUUUCU 5 381 382 AUGUGAAUGCAGACCAAAGAAAG AL-DP-4004 S634 5 GUGAAUGCAGACCAAAGAAAG 3 +++ ++ AS 635 3 UACACUUACGUCUGGUUUCUUUC 5AL-DP-4117 S 636 5 GUGAAUGCAGACCAAAGAAAG 3 +++ NA AS 6373 CACUUACGUCUGGUUUCUUUC 5 AL-DP-4016 S 638 5 GUGAAUGCAGACCAAAGAATT 3 ++++++ AS 639 3 TTCACUUACGUCUGGUUUCUU 5 AL-DP-4121 S 6405 GUGAAUGCAGACCAAAGAA 3 ++ NA AS 641 3 CACUUACGUCUGGUUUCUU 5 383 384GUGAAUGCAGACCAAAGAAAGAU AL-DP-4005 S 642 5 GAAUGCAGACCAAAGAAAGAU 3 +++++ AS 643 3 CACUUACGUCUGGUUUCUUUCUA 5 AL-DP-4053 S 6445 GAAUGCAGACCAAAGAAAGTT 3 +++ ++ AS 645 3 TTCUUACGUCUGGUUUCUUUC 5

TABLE 3 Phosphorothioate stabilized siRNA Molecules aremodified versions of AL-DP-4014. ORF SEQ Posi- Aln ID Effi- tionDuplex # Duplex Sequence NOs cacy 319 ALN-DP- 5′-G*C*GGAUCAAACCU 646 +++4127 CACCA*A*dT*dT-3′ 3′-dT*dT*C*GCCUAGU 647 UUGGAGUGG*U*U-5′ 319ALN-DP- 5′-G*C*GGAUCAAACCU 648 +++ 4128 C*ACC*A*A*dT*dT-3′3′-dT*dT*CGCCUAGUU 649 UGGAGUGGU*U-5′ 319 ALN-DP- 5′-G*C*GGAUCAAACCU 650+++ 4129 C*ACC*A*A*dT*dT-3′ 3′-dT*dT*C*GCCUAGU 651 UUGGAGUGG*U*U-5′*indicates the position of a phosphorothioate group

TABLE 4 In vitro efficacy of Modified AL-DP-4094 series Effi-5′-sense strand-3′ SEQ ID SiRNA cacy 3′-antisense strand-5′ NOsAL-DP-4198 AL4554 +++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4165 AL4554 +++5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4558′3-GsU_(OMe)C_(OMe)GUGUAUCCUCUCUACUGAsA-′5 654 AL-DP-4166 AL4554 +++5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4559′3-GsU_(OMe)C_(OMe)GUGUAU_(OMe)CCUCUCUACUGAsA-′5 655 AL-DP-4167 AL4554+++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4560′3-GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 656AL-DP-4168 AL4554 +++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4561′3-GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 657AL-DP-4169 AL4554 +++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4562′3-GsU_(OMe)dCGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAdCUCGAA-′5 658 AL-DP-4170AL4555 +++ 5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4171 AL4555 +++5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4558′3-GsU_(OMe)C_(OMe)GUGUAUCCUCUCUACUGAsA-′5 654 AL-DP-4172 AL4555 +++5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4559′3-GsU_(OMe)C_(OMe)GUGUAU_(OMe)CCUCUCUACUGAsA-′5 655 AL-DP-4173 AL4555+++ 5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4560′3-GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 656AL-DP-4174 AL4555 +++ 5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4561′3-GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 657AL-DP-4175 AL4555 +++ 5′-GsCACAU_(2′OMe)AGGAGAGAUGAGCUsU-3′ 659 AL4562′3-GsU_(OMe)dCGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAdCUCGAA-′5 658 AL-DP-4176AL4556 +++ 5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′660 AL4557 ′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4177 AL4556 +++5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660 AL4558′3-GsU_(OMe)C_(OMe)GUGUAUCCUCUCUACUGAsA-′5 654 AL-DP-4178 AL4556 +++5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660 AL4559′3-GsU_(OMe)C_(OMe)GUGUAU_(OMe)CCUCUCUACUGAsA-′5 655 AL-DP-4179 AL4556+++ 5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660AL4560 ′3-GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 656AL-DP-4180 AL4556 +++5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660 AL4561′3-GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 657AL-DP-4181 AL4556 +++5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660 AL4562′3-GsU_(OMe)dCGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAdCUCGAA-′5 658 AL-DP-4220AL2780 +++ 5′-GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′1085 AL2781′3-GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAsA-′5 995AL-DP-4182 AL4563 +++5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4183 AL4563 +++5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4558′3-GsU_(OMe)C_(OMe)GUGUAUCCUCUCUACUGAsA-′5 654 AL-DP-4184 AL4563 +++5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4559′3-GsU_(OMe)C_(OMe)GUGUAU_(OMe)CCUCUCUACUGAsA-′5 655 AL-DP-4185 AL4563+++ 5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4560′3-GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 656AL-DP-4186 AL4563 +++5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4561′3-GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAA-′5 657AL-DP-4187 AL4563 +++5′-G dC A dC AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 661 AL4562′3 -GsU_(OMe)dCGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAdCUCGAA-′5 658 AL-DP-4188AL4564 +++ 5′-GsCACAU_(F)AGGAGAGAUGAGCUsU-3′ 662 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4189 AL4565 +++5′-GC_(F)AC_(F)AU_(F)AGGAGAGAU_(F)GAGCU_(F)sU-3′ 663 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4190 AL4566 +++5′-GC_(F)AC_(F)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 664 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4191 AL4567 +++5′-GC_(OMe)AC_(OMe)AU_(F)AGGAGAGAU_(F)GAGCU_(F)sU-3′ 665 AL4557′3-GsUCGUGUAUCCUCUCUACUCGAsA-′5 653 AL-DP-4192 AL4554 +++5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4568′3-GsU_(F)CGU_(F)GU_(F)AU_(F)CCUCUCUAC_(F)UCGAA-′5 666 AL-DP-4193 AL4554+++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4569′3-GsU_(F)CGU_(F)GU_(F)AU_(F)CCUCUCUAC_(OMe)UCGAA-′5 667 AL-DP-4194AL4554 +++ 5′-GsCACAUAGGAGAGAUGAGCUsU-3′ 652 AL4570′3-GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(F)UCGAA-′5 668AL-DP-4197 AL4556 ND5′-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 660 AL4568′3-GsU_(F)CGU_(F)GU_(F)AU_(F)CCUCUCUAC_(F)UCGAA-′5 666 AL-DP-4221 AL2780+++ 5′-GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU-3′ 669AL2782 ′3-GsU_(F)CGU_(F)GU_(F)AU_(F)CCUCUCUAC_(F)UCGAsA-′5 670“Atugen Design” based on single overhang AL-DP-4195 AL4571 +5′-GcAcAuAgGaGaGaUgAgCusU-3′ 1089 AL4572 ′3-gsUcGuGuAuCcUcUcUaCuCgAa-′5672 d deoxynucleotide _(OMe)2′O-Methyl _(F)2′Fluoro s phosphorothioatelinkage N Mismatches in scrambled controls

TABLE 5 In vitro efficacy of siRNAs in HeLa cells Unmodified strandEffi- 5′-sense strand-3′ SEQ ID siRNA parent # cacy3′-antisense strand-5′ NOs AL-DP- AL-DP- AL2732 +++ 5′CsAAGUGGUCCCAGGCUGCATsT 3′ 673 4374 4055 AL2740 3′TsTGUUCACCAGGGUCCGACGsU 5′ 674 AL-DP- AL-DP- AL2728 +++ 5′GsGACAUCUUCCAGGAGUACTsT 3′ 675 4375 4015 AL2730 3′TsTCCUGUAGAAGGUCCUCAUsG 5′ 676 AL-DP- AL-DP- AL2963 +++ 5′C_(OMe)C_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)C_(OMe)AGGC_(OMe)U_(OMe)GC_(OMe)TsT3′677 4379 4088 AL2964 3′TsTGGU_(F)U_(F)C_(F)AC_(F)C_(F)AGGGU_(F)C_(F)C_(F)GAC_(F)G 5′ 678 AL-DP-AL-DP- AL2966 +++ 5′GC_(OMe)GGAU_(OMe)C_(OMe)AAAC_(OMe)C_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AATsT 3′679 4380 4014 AL2967 3′′TsTC_(F)GC_(F)C_(F)U_(F)AGU_(F)U_(F)U_(F)GGAGU_(F)GGU_(F)U_(F)5' 680AL-DP- AL-DP- AL2712 +++ 5′ GsUGAAUGCAGACCAAAGAAAsG 3′ 681 4219 4004AL2720 3′ UsACACUUACGUCUGGUUUCUUUsC 5′ 682 AL-DP- AL-DP- AL2281 − 5′GsCsGGAACAAUCCUGACCAsAsTsT 3′ 683 4140 4014 AL2282 3′TsTCGCCUUGUUAGGACUGGsUsU 5′ 684 _(OMe)2′O-Methyl _(F)2′Fluoro sphosphorothioate linkage N Mismatches in scrambled controls

TABLE 6Oligonucleotides with phosphorothioate, 2′-O-methyl, and 2′-fluoro modificationsand in vitro efficacy against VEGF. in PARENT SEQ vitro AL-DP-# AL-AL-SQ ID Effi- and ORF DP-# # DUPLEX SEQUENCE AND MODIFICATIONS NOs cacyMass 4103 4034 CCACCAUGCCAAGUGGUCCdTdT 685 ++ ORF 52 4132dTdTGGUGGUACGGUUCACCAGG 686 4222 2510CsC_(OMe)sAC_(OMe)CA_(OMe)UG_(OMe)CC_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)sCsdTsdT687 − 6810.3 2511dTsdTsGsG_(OMe)UG_(OMe)GU_(OMe)AC_(OMe)GG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AsG_(OMe)sG688 6947.4 4223 2540C_(OMe)sCsA_(OMe)CC_(OMe)AU_(OMe)GC_(OMe)CA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)Cs_(OMe)sdTsdT689 − 6824.3 2541dTsdTsG_(OMe)sGU_(OMe)GG_(OMe)UA_(OMe)CG_(OMe)GU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)sGsG_(OMe)690 6961.4 4224 2510CsC_(OMe)sAC_(OMe)CA_(OMe)UG_(OMe)CC_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)sCsdTsdT687 +/− 6810.3 2541dTsdTsG_(OMe)sGU_(OMe)GG_(OMe)UA_(OMe)CG_(OMe)GU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)sGsG_(OMe)690 6961.4 4225 2540C_(OMe)sCsA_(OMe)CC_(OMe)AU_(OMe)GC_(OMe)CA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)CsC_(OMe)sdTsdT689 − 6824.3 2511dTsdTsGsG_(OMe)UG_(OMe)GU_(OMe)AC_(OMe)GG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AsG_(OMe)sG688 6947.4 4226 2570C_(OMe)sC_(OMe)AC_(OMe)C_(OMe)AU_(OMe)GC_(OMe)C_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)dTsdT691 − 6790.4 2571dTsdTGGU_(OMe)GGU_(OMe)AC_(OMe)GGU_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AGsG692 6885.4 4227 2600CsC_(OMe)ACC_(OMe)AU_(OMe)GCC_(OMe)AAGU_(OMe)GGUCCdTsdT 693 − 6706.22601 dTsdTGGU_(OMe)GGU_(OMe)AC_(OMe)GGU_(OMe)UCAC_(OMe)CAGsG 694 6843.34228 2570C_(OMe)sC_(OMe)AC_(OMe)C_(OMe)AU_(OMe)GC_(OMe)C_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)dTsdT691 − 6790.4 2631dTsdTGGU_(F)GGU_(F)AC_(F)GGU_(F)U_(F)C_(F)AC_(F)C_(F)AGsG 695 6789.14229 2600 CsC_(OMe)ACC_(OMe)AU_(OMe)GCC_(OMe)AAGU_(OMe)GGUCCdTsdT 693 +6706.2 2661 dTsdTGGU_(F)GGU_(F)AC_(F)GGU_(F)UCAC_(F)CAGsG 696 6783.14088 4042 CCAAGUGGUCCCAGGCUGCdTdT 697 +++ ORF 60 4140dTdTGGUUCACCAGGGUCCGACG 698 4230 2512CsC_(OMe)sAA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)CC_(OMe)AG_(OMe)GC_(OMe)UG_(OMe)sCsdTsdT699 − 6866.3 2513dTsdTsGsG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AG_(OMe)GG_(OMe)UC_(OMe)CG_(OMe)AsC_(OMe)sG_(OMe)700 6906.4 4231 2542C_(OMe)sCsA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)CC_(OMe)CA_(OMe)GG_(OMe)CU_(OMe)GsC_(OMe)sdTsdT701 − 6880.4 2543dTsdTsG_(OMe)sGU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)GG_(OMe)GU_(OMe)CC_(OMe)GA_(OMe)sCsG_(OMe)702 6920.4 4232 2512CsC_(OMe)sAA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)CC_(OMe)AG_(OMe)GC_(OMe)UG_(OMe)sCsdTsdT699 − 6866.3 2543dTsdTsG_(OMe)sGU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)GG_(OMe)GU_(OMe)CC_(OMe)GA_(OMe)sCsG_(OMe)702 6920.4 4233 2542C_(OMe)sCsA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)CC_(OMe)CA_(OMe)GG_(OMe)CU_(OMe)GsC_(OMe)sdTsdT701 − 6880.4 2513dTsdTsGsG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AG_(OMe)GG_(OMe)UC_(OMe)CG_(OMe)AsC_(OMe)sG700 6906.4 4234 2572C_(OMe)sC_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)C_(OMe)AGGC_(OMe)U_(OMe)GC_(OMe)dTsdT703 − 6832.4 2573dTsdTGGU_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AGGGU_(OMe)C_(OMe)C_(OMe)GAC_(OMe)sG704 6858.4 4235 2602 CsC_(OMe)AAGU_(OMe)GGUCCC_(OMe)AGGCU_(OMe)GCdTsdT705 + 6748.2 2603 dTsdTGGUUCAC_(OMe)CAGGGU_(OMe)CCGAC_(OMe)sG 706 6788.24236 2572C_(OMe)sC_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)C_(OMe)AGGC_(OMe)U_(OMe)GC_(OMe)dTsdT703 +++ 6832.4 2633dTsdTGGU_(F)U_(F)C_(F)AC_(F)C_(F)AGGGU_(F)C_(F)C_(F)GAC_(OMe)sG 7076750.1 4237 2602 CsC_(OMe)AAGU_(OMe)GGUCCC_(OMe)AGGCU_(OMe)GCdTsdT 705+++ 6748.2 2663 dTsdTGGU_(F)UCAC_(F)CAGGGU_(F)CCGAC_(F)sG 708 6740.14055 4043 CAAGUGGUCCCAGGCUGCAdTdT 709 +++ ORF 61 4141dTdTGUUCACCAGGGUCCGACGU 710 4358 2736CA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)CC_(OMe)CA_(OMe)GG_(OMe)CU_(OMe)GC_(OMe)AdTsdT711 − 2744dTsdTGU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)GG_(OMe)GU_(OMe)CC_(OMe)GA_(OMe)CG_(OMe)U712 4359 2737C_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)CC_(OMe)AG_(OMe)GC_(OMe)UG_(OMe)CA_(OMe)dTsdT713 − 2745dTsdTG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AG_(OMe)GG_(OMe)UC_(OMe)CG_(OMe)AC_(OMe)GU_(OMe)714 4360 2736CA_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)CC_(OMe)CA_(OMe)GG_(OMe)CU_(OMe)GC_(OMe)AdTsdT711 − 2745dTsdTG_(OMe)UU_(OMe)CA_(OMe)CC_(OMe)AG_(OMe)GG_(OMe)UC_(OMe)CG_(OMe)AC_(OMe)GU_(OMe)714 4361 2737C_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)UC_(OMe)CC_(OMe)AG_(OMe)GC_(OMe)UG_(OMe)CA_(OMe)dTsdT713 − 2744dTsdTGU_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)GG_(OMe)GU_(OMe)CC_(OMe)GA_(OMe)CG_(OMe)U712 4362 2735C_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)C_(OMe)AGGC_(OMe)U_(OMe)GC_(OMe)AdTsdT715 − 2743dTsdTGU_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AGGGU_(OMe)C_(OMe)C_(OMe)GAC_(OMe)GU_(OMe)716 4363 2734 C_(OMe)AAGU_(OMe)GGUCCC_(OMe)AGGCU_(OMe)GC_(OMe)AdTsdT 717− 2742 dTsdTGU_(OMe)UCAC_(OMe)CAGGGU_(OMe)CCGAC_(OMe)GU_(OMe) 718 43642735C_(OMe)AAGU_(OMe)GGU_(OMe)C_(OMe)C_(OMe)C_(OMe)AGGC_(OMe)U_(OMe)GC_(OMe)AdTsdT715 −? 2747dTsdTGU_(F)U_(F)C_(F)AC_(F)C_(F)AGGGU_(F)C_(F)C_(F)GAC_(F)GU_(F) 7194365 2734 C_(OMe)AAGU_(OMe)GGUCCC_(OMe)AGGCU_(OMe)GC_(OMe)AdTsdT 717 +++2746 dTsdTGU_(F)UCAC_(F)CAGGGU_(F)CCGAC_(F)GU_(F) 720 4109 4003AGAAUCAUCACGAAGUGGUdTdT 721 ++ ORF 102 4070 dTdTUCUUAGUAGUGCUUCACCA 7224238 2514AsG_(OMe)sAA_(OMe)UC_(OMe)AUCA_(OMe)CG_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)sUsdTsdT723 − 6923.4 2515dTsdTsUsC_(OMe)UU_(OMe)AG_(OMe)UA_(OMe)GU_(OMe)GC_(OMe)UU_(OMe)CA_(OMe)CsC_(OMe)sA724 6774.2 4239 2544A_(OMe)sGsA_(OMe)AU_(OMe)CA_(OMe)UC_(OMe)AC_(OMe)GA_(OMe)AG_(OMe)UG_(OMe)GsU_(OMe)sdTsdT725 − 6937.4 2545dTsdTsU_(OMe)sCU_(OMe)UA_(OMe)GU_(OMe)AG_(OMe)UG_(OMe)CU_(OMe)UC_(OMe)AC_(OMe)sCsA_(OMe)726 6788.3 4240 2514AsG_(OMe)sAA_(OMe)UC_(OMe)AUCA_(OMe)CG_(OMe)AA_(OMe)GU_(OMe)GG_(OMe)sUsdTsdT723 − 6923.4 2545dTsdTsU_(OMe)sCU_(OMe)UA_(OMe)GU_(OMe)AG_(OMe)UG_(OMe)CU_(OMe)UC_(OMe)AC_(OMe)sCsA_(OMe)726 6788.3 4241 2544A_(OMe)sGsA_(OMe)AU_(OMe)CA_(OMe)UC_(OMe)AC_(OMe)GA_(OMe)AG_(OMe)UG_(OMe)GsU_(OMe)sdTsdT725 − 6937.4 2515dTsdTsUsC_(OMe)UU_(OMe)AG_(OMe)UA_(OMe)GU_(OMe)GC_(OMe)UU_(OMe)CA_(OMe)CsC_(OMe)sA724 6774.2 4242 2574A_(OMe)sGAAU_(OMe)C_(OMe)AU_(OMe)C_(OMe)AC_(OMe)GAAGU_(OMe)GGU_(OMe)dTsdT727 − 6847.4 2575dTsdTU_(OMe)C_(OMe)U_(OMe)U_(OMe)AGU_(OMe)AGU_(OMe)GC_(OMe)U_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)sA728 6768.3 4243 2604 AsGAAUC_(OMe)AUC_(OMe)ACGAAGU_(OMe)GGUdTsdT 729 −6791.2 2605 dTsdTUCUUAGU_(OMe)AGU_(OMe)GCUUCAC_(OMe)CsA 730 6642.1 42442574A_(OMe)sGAAU_(OMe)C_(OMe)AU_(OMe)C_(OMe)AC_(OMe)GAAGU_(OMe)GGU_(OMe)dTsdT727 + 6847.4 2635dTsdTU_(F)C_(F)U_(F)U_(F)AGU_(F)AGU_(F)GC_(OMe)U_(F)U_(F)C_(F)AC_(F)C_(F)sA731 6624.0 4245 2604 AsGAAUC_(OMe)AUC_(OMe)ACGAAGU_(OMe)GGUdTsdT 729 ++6791.2 2665 dTsdTUCUUAGU_(F)AGU_(F)GCUUCAC_(F)CsA 732 6606.0 4111 4007UGGAUGUCUAUCAGCGCAGdTdT 733 +++ ORF 4074 dTdTACCUACAGAUAGUCGCGUC 7344246 2516UsG_(OMe)sGA_(OMe)UG_(OMe)UC_(OMe)UA_(OMe)UC_(OMe)AG_(OMe)CG_(OMe)CA_(OMe)sGsdTsdT735 − 6892.3 2517dTsdTsAsC_(OMe)CU_(OMe)AC_(OMe)AG_(OMe)AU_(OMe)AG_(OMe)UC_(OMe)GC_(OMe)GsU_(OMe)sC736 6835.3 4247 2546U_(OMe)sGsG_(OMe)AU_(OMe)GU_(OMe)CU_(OMe)AU_(OMe)CA_(OMe)GC_(OMe)GC_(OMe)AsG_(OMe)sdTsdT737 − 6906.4 2547dTsdTsA_(OMe)sCC_(OMe)UA_(OMe)CA_(OMe)GA_(OMe)UA_(OMe)GU_(OMe)CG_(OMe)CG_(OMe)sUsC_(OMe)738 6849.4 4248 2516UsG_(OMe)sGA_(OMe)UG_(OMe)UC_(OMe)UA_(OMe)UC_(OMe)AG_(OMe)CG_(OMe)CA_(OMe)sGsdTsdT735 − 6892.3 2547dTsdTsA_(OMe)sCC_(OMe)UA_(OMe)CA_(OMe)GA_(OMe)UA_(OMe)GU_(OMe)CG_(OMe)CG_(OMe)sUsC_(OMe)738 6849.4 4249 2546U_(OMe)sGsG_(OMe)AU_(OMe)GU_(OMe)CU_(OMe)AU_(OMe)CA_(OMe)GC_(OMe)GC_(OMe)AsG_(OMe)sdTsdT737 − 6906.4 2517dTsdTsAsC_(OMe)CU_(OMe)AC_(OMe)AG_(OMe)AU_(OMe)AG_(OMe)UC_(OMe)GC_(OMe)GsU_(OMe)sC736 6835.3 4250 2576U_(OMe)sGGAU_(OMe)GUC_(OMe)U_(OMe)AU_(OMe)C_(OMe)AGC_(OMe)GC_(OMe)A_(OMe)GdTsdT739 − 6844.3 2577dTsdTAC_(OMe)C_(OMe)U_(OMe)AC_(OMe)AGAU_(OMe)AGU_(OMe)C_(OMe)GC_(OMe)GU_(OMe)sC_(OMe)740 6801.4 4251 2606 UsGGAU_(OMe)GUCU_(OMe)AUC_(OMe)AGCGC_(OMe)AGdTsdT741 − 6788.2 2607dTsdTAC_(OMe)CUAC_(OMe)AGAU_(OMe)AGU_(OMe)CGCGU_(OMe)sC 742 6731.2 42522576U_(OMe)sGGAU_(OMe)GUC_(OMe)U_(OMe)AU_(OMe)C_(OMe)AGC_(OMe)GC_(OMe)A_(OMe)GdTsdT739 + 6844.3 2637dTsdTAC_(F)C_(F)U_(F)AC_(F)AGAU_(OMe)AGU_(F)C_(F)GC_(F)GU_(F)sC_(F) 7436681.1 4253 2606 UsGGAU_(OMe)GUCU_(OMe)AUC_(OMe)AGCGC_(OMe)AGdTsdT 744+++ 6788.2 2667 dTsdTAC_(F)CUAC_(F)AGAU_(F)AGU_(F)CGCGU_(F)sC 745 6671.14028 ++ 4014 UACUGCCAUCCAAUCGAGAdTdT 746 ORF 149 4081dTdTAUGACGGUAGGUUAGCUCU 747 4254 2518UsA_(OMe)sCU_(OMe)GC_(OMe)CA_(OMe)UC_(OMe)CA_(OMe)AU_(OMe)CG_(OMe)AG_(OMe)sAsdTsdT748 No 6819.3 2519dTsdTsAsU_(OMe)GA_(OMe)CG_(OMe)GU_(OMe)AG_(OMe)GU_(OMe)UA_(OMe)GC_(OMe)UsC_(OMe)sU749 data 6893.3 4255 2548U_(OMe)sAsC_(OMe)UG_(OMe)CC_(OMe)AU_(OMe)CC_(OMe)AA_(OMe)UC_(OMe)GA_(OMe)GsA_(OMe)sdTsdT750 No 6833.4 2549dTsdTsA_(OMe)sUG_(OMe)AC_(OMe)GG_(OMe)UA_(OMe)GG_(OMe)UU_(OMe)AG_(OMe)CU_(OMe)sCsU_(OMe)751 data 6907.4 4256 2518UsA_(OMe)sCU_(OMe)GC_(OMe)CA_(OMe)UC_(OMe)CA_(OMe)AU_(OMe)CG_(OMe)AG_(OMe)sAsdTsdT748 No 6819.3 2549dTsdTsA_(OMe)sUG_(OMe)AC_(OMe)GG_(OMe)UA_(OMe)GG_(OMe)UU_(OMe)AG_(OMe)CU_(OMe)sCsU_(OMe)751 data 6907.4 4257 2548U_(OMe)sAsC_(OMe)UG_(OMe)CC_(OMe)AU_(OMe)CC_(OMe)AA_(OMe)UC_(OMe)GA_(OMe)GsA_(OMe)sdTsdT750 No 6833.4 2519dTsdTsAsU_(OMe)GA_(OMe)CG_(OMe)GU_(OMe)AG_(OMe)GU_(OMe)UA_(OMe)GC_(OMe)UsC_(OMe)sU749 data 6893.3 4258 2578U_(OMe)sAC_(OMe)U_(OMe)GC_(OMe)C_(OMe)AU_(OMe)C_(OMe)C_(OMe)AAU_(OMe)C_(OMe)GAGAdTsdT752 − 6785.4 2579dTsdTAU_(OMe)GAC_(OMe)GGU_(OMe)AGGU_(OMe)U_(OMe)AGC_(OMe)U_(OMe)C_(OMe)sU_(OMe)753 6845.3 4259 2608 UsACU_(OMe)GCC_(OMe)AUCC_(OMe)AAUCGAGAdTsdT 754 ++6701.2 2609 dTsdTAU_(OMe)GAC_(OMe)GGU_(OMe)AGGU_(OMe)UAGCUCsU 755 6775.24260 2578U_(OMe)sAC_(OMe)U_(OMe)GC_(OMe)C_(OMe)AU_(OMe)C_(OMe)C_(OMe)AAU_(OMe)C_(OMe)GAGAdTsdT752 + 6785.4 2639dTsdTAU_(F)GAC_(F)GGU_(F)AGGU_(F)U_(F)AGC_(F)U_(F)C_(F)sU_(F) 756 6721.14261 2608 UsACU_(OMe)GCC_(OMe)AUCC_(OMe)AAUCGAGAdTsdT 754 + 6701.2 2669dTsdTAU_(F)GAC_(F)GGU_(F)AGGU_(F)UAGCUCsU 757 6727.1 4060 4061CCCUGGUGGACAUCUUCCAdTdT 758 +++ ORF 168 4159 dTdTGGGACCACCUGUAGAAGGU 7594262 2520CsC_(OMe)sCU_(OMe)GG_(OMe)UG_(OMe)GA_(OMe)CA_(OMe)UC_(OMe)UU_(OMe)CC_(OMe)sAsdTsdT760 − 6788.3 2521dTsdTsGsG_(OMe)GA_(OMe)CC_(OMe)AC_(OMe)CU_(OMe)GU_(OMe)AG_(OMe)AA_(OMe)GsG_(OMe)sU761 6954.4 4263 2550C_(OMe)sCsC_(OMe)UG_(OMe)GU_(OMe)GG_(OMe)AC_(OMe)AU_(OMe)CU_(OMe)UC_(OMe)CsA_(OMe)sdTsdT762 − 6802.3 2551dTsdTsG_(OMe)sGG_(OMe)AC_(OMe)CA_(OMe)CC_(OMe)UG_(OMe)UA_(OMe)GA_(OMe)AG_(OMe)sGsU_(OMe)763 6968.5 4264 2520CsC_(OMe)sCU_(OMe)GG_(OMe)UG_(OMe)GA_(OMe)CA_(OMe)UC_(OMe)UU_(OMe)CC_(OMe)sAsdTsdT760 − 6788.3 2551dTsdTsG_(OMe)sGG_(OMe)AC_(OMe)CA_(OMe)CC_(OMe)UG_(OMe)UA_(OMe)GA_(OMe)AG_(OMe)sGsU_(OMe)763 6968.5 4265 2550C_(OMe)sCsC_(OMe)UG_(OMe)GU_(OMe)GG_(OMe)AC_(OMe)AU_(OMe)CU_(OMe)UC_(OMe)CsA_(OMe)sdTsdT762 − 6802.3 2521dTsdTsGsG_(OMe)GA_(OMe)CC_(OMe)AC_(OMe)CU_(OMe)GU_(OMe)AG_(OMe)AA_(OMe)GsG_(OMe)sU761 6954.4 4266 2580C_(OMe)sC_(OMe)C_(OMe)U_(OMe)GGU_(OMe)GGAC_(OMe)AU_(OMe)C_(OMe)U_(OMe)U_(OMe)C_(OMe)C_(OMe)AdTsdT764 − 6782.3 2581dTsdTGGGAC_(OMe)C_(OMe)AC_(OMe)C_(OMe)U_(OMe)GU_(OMe)AGAAGGsU_(OMe) 7656878.4 4267 2610 CsCCU_(OMe)GGU_(OMe)GGAC_(OMe)AUCUUCC_(OMe)AdTsdT 766 +6670.1 2611 dtsdTGGGAC_(OMe)CAC_(OMe)CUGU_(OMe)AGGsU_(OMe) 767 6926.34268 2580C_(OMe)sC_(OMe)C_(OMe)U_(OMe)GGU_(OMe)GGAC_(OMe)AU_(OMe)C_(OMe)U_(OMe)U_(OMe)C_(OMe)C_(OMe)AdTsdT764 ++ 6782.3 2641 dTsdTGGGAC_(F)C_(F)AC_(F)C_(F)U_(F)GU_(F)AGAAGGsU_(F)768 6778.2 4269 2610 CsCCU_(OMe)GGU_(OMe)GGAC_(OMe)AUCUUCC_(OMe)AdTsdT766 + 6670.1 2671 dTsdTGGGAC_(F)CAC_(F)CUGU_(F)AGAAGGsU_(F) 769 6772.24015 4066 GGACAUCUUCCAGGAGUACdTdT 770 +++ ORF 175 4164dTdTCCUGUAGAAGGUCCUCAUG 771 4270 2522GsG_(OMe)sAC_(OMe)AU_(OMe)CU_(OMe)UC_(OMe)CA_(OMe)GG_(OMe)AG_(OMe)UA_(OMe)sCsdTsdT772 − 6875.4 2523dTsdTsCsC_(OMe)UG_(OMe)UA_(OMe)GA_(OMe)AG_(OMe)GU_(OMe)CC_(OMe)UC_(OMe)AsU_(OMe)sG773 6852.3 4271 2552G_(OMe)sGsA_(OMe)CA_(OMe)UC_(OMe)UU_(OMe)CC_(OMe)AG_(OMe)GA_(OMe)GU_(OMe)AsC_(OMe)sdTsdT774 − 6889.4 2553dTsdTsC_(OMe)sCU_(OMe)GU_(OMe)AG_(OMe)AA_(OMe)GG_(OMe)UC_(OMe)CU_(OMe)CA_(OMe)sUsG_(OMe)775 6866.3 4272 2522GsG_(OMe)sAC_(OMe)AU_(OMe)CU_(OMe)UC_(OMe)CA_(OMe)GG_(OMe)AG_(OMe)UA_(OMe)sCsdTsdT772 − 6875.4 2553dTsdTsC_(OMe)sCU_(OMe)GU_(OMe)AG_(OMe)AA_(OMe)GG_(OMe)UC_(OMe)CU_(OMe)CA_(OMe)sUsG_(OMe)775 6866.3 4273 2552G_(OMe)sGsA_(OMe)CA_(OMe)UC_(OMe)UU_(OMe)CC_(OMe)AG_(OMe)GA_(OMe)GU_(OMe)AsC_(OMe)sdTsdT774 − 6889.4 2523dTsdTsCsC_(OMe)UG_(OMe)UA_(OMe)GA_(OMe)AG_(OMe)GU_(OMe)CC_(OMe)UC_(OMe)AsU_(OMe)sG773 6852.3 4274 2582G_(OMe)sGAC_(OMe)AU_(OMe)C_(OMe)U_(OMe)U_(OMe)C_(OMe)C_(OMe)AGGAGU_(OMe)AC_(OMe)dTsdT776 − 6827.4 2583dTsdTC_(OMe)C_(OMe)U_(OMe)GU_(OMe)AGAAGGU_(OMe)C_(OMe)C_(OMe)U_(OMe)C_(OMe)AU_(OMe)sG777 6818.3 4275 2612 GsGAC_(OMe)AUCUUCC_(OMe)AGGAGU_(OMe)ACdTsdT 778 −6743.2 2613 dTsdTCCUGU_(OMe)AGAAGGU_(OMe)CCUCAU_(OMe)sG 779 6720.1 42762582G_(OMe)sGAC_(OMe)AU_(OMe)C_(OMe)U_(OMe)U_(OMe)C_(OMe)C_(OMe)AGGAGU_(OMe)AC_(OMe)dTsdT776 − 6827.4 2643dTsdTC_(F)C_(F)U_(F)GU_(F)AGAAGGU_(F)C_(F)C_(F)U_(F)C_(F)AU_(F)sG 7806698.0 4277 2612 GsGAC_(OMe)AUCUUCC_(OMe)AGGAGU_(OMe)ACdTsdT 778 +++6743.2 2673 dTsdTCCUGU_(F)AGAAGGU_(F)CCUCAU_(F)sG 781 6684.0 4032 4025UACCCUGAUGAGAUCGAGUdTdT 782 +++ ORF 191 4092 dTdTAUGGGACUACUCUAGCUCA 7834278 2524UsA_(OMe)sCC_(OMe)CU_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)AU_(OMe)CG_(OMe)AG_(OMe)sUsdTsdT784 + 6876.3 2525dTsdTsAsU_(OMe)GG_(OMe)GA_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)UA_(OMe)GC_(OMe)UsC_(OMe)sA785 6836.3 4279 2554U_(OMe)sAsC_(OMe)CC_(OMe)UG_(OMe)AU_(OMe)GA_(OMe)GA_(OMe)UC_(OMe)GA_(OMe)GsU_(OMe)sdTsdT786 + 6890.4 2555dTsdTsA_(OMe)sUG_(OMe)GG_(OMe)AC_(OMe)UA_(OMe)CU_(OMe)CU_(OMe)AG_(OMe)CU_(OMe)sCsA_(OMe)787 6850.3 4280 2524UsA_(OMe)sCC_(OMe)CU_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)AU_(OMe)CG_(OMe)AG_(OMe)sUsdTsdT784 ++ 6876.3 2555dTsdTsA_(OMe)sUG_(OMe)GG_(OMe)AC_(OMe)UA_(OMe)CU_(OMe)CU_(OMe)AG_(OMe)CU_(OMe)sCsA_(OMe)787 6850.3 4281 2554U_(OMe)sAsC_(OMe)CC_(OMe)UG_(OMe)AU_(OMe)GA_(OMe)GA_(OMe)UC_(OMe)GA_(OMe)GsU_(OMe)sdTsdT786 − 6890.4 2525dTsdTsAsU_(OMe)GG_(OMe)GA_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)UA_(OMe)GC_(OMe)UsC_(OMe)sA785 6836.3 4282 2584U_(OMe)sAC_(OMe)C_(OMe)C_(OMe)U_(OMe)GAU_(OMe)GAGAU_(OMe)C_(OMe)GAGU_(OMe)dTsdT788 − 6828.3 2585 dTsdTAU_(OMe)GGGACU_(OMe)ACUCU_(OMe)AGCUC_(OMe)sA 7896802.3 4283 2614 UsACCCU_(OMe)GAU_(OMe)GAGAUCGAGUdTsdT 790 +++ 6744.22615 dTsdTAU_(OMe)GGGAC_(OMe)UAC_(OMe)UCUAGCUCsA 791 6704.1 4284 2584U_(OMe)sAC_(OMe)C_(OMe)C_(OMe)U_(OMe)GAU_(OMe)GAGAU_(OMe)C_(OMe)GAGU_(OMe)dTsdT788 +++ 6828.3 2645dTsdTAU_(F)GGGAC_(F)U_(F)AC_(F)U_(F)C_(F)U_(F)AGC_(F)U_(F)C_(F)sA 7926682.0 4285 2614 UsACCCU_(OMe)GAU_(OMe)GAGAUCGAGUdTsdT 790 ++ 6744.22675 dTsdTAU_(F)GGGAC_(F)UAC_(F)UCUAGCUCsA 793 6668.0 4033 4026ACCAUGCAGAUUAUGCGGAdTdT 794 ++ ORF 305 4093 dTdTUGGUACGUCUAAUACGCCU 7954286 2526AsC_(OMe)sCA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)UU_(OMe)AU_(OMe)GC_(OMe)GG_(OMe)sAsdTsdT796 ++ 6899.4 2527dTsdTsUsG_(OMe)GU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)AA_(OMe)UA_(OMe)CG_(OMe)CsC_(OMe)sU797 6813.3 4287 2556A_(OMe)sCsC_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AU_(OMe)UA_(OMe)UG_(OMe)CG_(OMe)GsA_(OMe)sdTsdT798 + 6913.4 2557dTsdTsU_(OMe)sGG_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UA_(OMe)AU_(OMe)AC_(OMe)GC_(OMe)sCsU_(OMe)799 6827.3 4288 2526AsC_(OMe)sCA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)UU_(OMe)AU_(OMe)GC_(OMe)GG_(OMe)sAsdTsdT796 − 6899.4 2557dTsdTsU_(OMe)sGG_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UA_(OMe)AU_(OMe)AC_(OMe)GC_(OMe)sCsU_(OMe)799 6827.3 4289 2556A_(OMe)sCsC_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AU_(OMe)UA_(OMe)UG_(OMe)CG_(OMe)GsA_(OMe)sdTsdT798 − 6913.4 2527dTsdTsUsG_(OMe)GU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)AA_(OMe)UA_(OMe)CG_(OMe)CsC_(OMe)sU797 6813.3 4290 2586A_(OMe)sCC_(OMe)AU_(OMe)GC_(OMe)AGAU_(OMe)U_(OMe)AU_(OMe)GC_(OMe)GGAdTsdT800 − 6837.4 2587dTsdTU_(OMe)GGU_(OMe)AC_(OMe)GU_(OMe)C_(OMe)U_(OMe)AAU_(OMe)AC_(OMe)GC_(OMe)C_(OMe)sU_(OMe)801 6793.3 4291 2616AsCC_(OMe)AU_(OMe)GC_(OMe)AGAUU_(OMe)AU_(OMe)GCGGAdTsdT 802 − 6795.32617 dTsdTUGGU_(OMe)AC_(OMe)GU_(OMe)CUAAU_(OMe)AC_(OMe)GCCsU 803 6709.24292 2586A_(OMe)sCC_(OMe)AU_(OMe)GC_(OMe)AGAU_(OMe)U_(OMe)AU_(OMe)GC_(OMe)GGAdTsdT800 +++ 6837.4 2647dTsdTU_(F)GGU_(F)AC_(F)GU_(F)C_(F)U_(F)AAU_(F)A_(F)C_(F)GC_(F)C_(F)sU_(F)804 6645 4293 2616AsCC_(OMe)AU_(OMe)GC_(OMe)AGAUU_(OMe)AU_(OMe)GCGGAdTsdT 802 +++ 6795.32677 dTsdTUGGU_(F)AC_(F)GU_(F)CUAAU_(F)AC_(F)GCCsU 805 6649.0 4014 4112GCGGAUCAAACCUCACCAAdTdT 806 +++ ORF 319 4180 dTdTCGCCUAGUUUGGAGUGGUU 8074294 2528GsC_(OMe)sGG_(OMe)AU_(OMe)CA_(OMe)AA_(OMe)CC_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)sAsdTsdT808 + 6841.4 2529dTsdTsCsG_(OMe)CC_(OMe)UA_(OMe)GU_(OMe)UU_(OMe)GG_(OMe)AG_(OMe)UG_(OMe)GsU_(OMe)sU809 6886.3 4295 2558G_(OMe)sCsG_(OMe)GA_(OMe)UC_(OMe)AA_(OMe)AC_(OMe)CU_(OMe)CA_(OMe)CC_(OMe)AsA_(OMe)sdTsdT810 − 6855.4 2559dTsdTsC_(OMe)sGC_(OMe)CU_(OMe)AG_(OMe)UU_(OMe)UG_(OMe)GA_(OMe)GU_(OMe)GG_(OMe)sUsU_(OMe)811 6900.3 4296 2528GsC_(OMe)sGG_(OMe)AU_(OMe)CA_(OMe)AA_(OMe)CC_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)sAsdTsdT808 − 6841.4 2559dTsdTsC_(OMe)sGC_(OMe)CU_(OMe)AG_(OMe)UU_(OMe)UG_(OMe)GA_(OMe)GU_(OMe)GG_(OMe)sUsU_(OMe)811 6900.3 4297 2558G_(OMe)sCsG_(OMe)GA_(OMe)UC_(OMe)AA_(OMe)AC_(OMe)CU_(OMe)CA_(OMe)CC_(OMe)AsA_(OMe)sdTsdT810 − 6855.4 2529dTsdTsCsG_(OMe)CC_(OMe)UA_(OMe)GU_(OMe)UU_(OMe)GG_(OMe)AG_(OMe)UG_(OMe)GsU_(OMe)sU809 6886.3 4298 2588G_(OMe)sC_(OMe)GGAU_(OMe)C_(OMe)AAAC_(OMe)C_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AAdTsdT812 − 6793.4 2589dTsdTC_(OMe)GC_(OMe)C_(OMe)U_(OMe)AGU_(OMe)U_(OMe)U_(OMe)GGAGU_(OMe)GGU_(OMe)sU_(OMe)813 6852.3 4299 2618 GsCGGAUC_(OMe)AAACCUC_(OMe)ACC_(OMe)AAdTsdT 814 −6709.2 2619 dTsdTCGCCUAGU_(OMe)UUGGAGU_(OMe)GGU_(OMe)sU 815 6754.1 43002588G_(OMe)sC_(OMe)GGAU_(OMe)C_(OMe)AAAC_(OMe)C_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AAdTsdT812 + 6793.4 2649dTsdTC_(F)GC_(F)C_(F)U_(OMe)AGU_(F)U_(F)U_(F)GGAGU_(F)GGU_(F)sU_(F) 8166716.0 4301 2618 GsCGGAUC_(OMe)AAACCUC_(OMe)ACC_(OMe)AAdTsdT 814 +++6709.2 2679 dTsdTCGCCUAGU_(F)UUGGAGU_(F)GGU_(F)sU 817 6718.0 4123 4362ACCUCACCAAGGCCAGCACdTdT 818 ++ ORF 330 4363 dTdTUGGAGUGGUUCCGGUCGUG 8194302 2530AsC_(OMe)sCU_(OMe)CA_(OMe)CC_(OMe)AA_(OMe)GG_(OMe)CC_(OMe)AG_(OMe)CA_(OMe)sCsdTsdT820 + 6816.4 2531dTsdTsUsG_(OMe)GA_(OMe)GU_(OMe)GG_(OMe)UU_(OMe)CC_(OMe)GG_(OMe)UC_(OMe)GsU_(OMe)sG821 6941.3 4303 2560A_(OMe)sCsC_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)AG_(OMe)GC_(OMe)CA_(OMe)GC_(OMe)AsC_(OMe)sdTsdT822 − 6830.4 2561dTsdTsU_(OMe)sGG_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)UC_(OMe)CG_(OMe)GU_(OMe)CG_(OMe)sUsG_(OMe)823 6955.4 4304 2530AsC_(OMe)sCU_(OMe)CA_(OMe)CC_(OMe)AA_(OMe)GG_(OMe)CC_(OMe)AG_(OMe)CA_(OMe)sCsdTsdT820 − 6816.4 2561dTsdTsU_(OMe)sGG_(OMe)AG_(OMe)UG_(OMe)GU_(OMe)UC_(OMe)CG_(OMe)GU_(OMe)CG_(OMe)sUsG_(OMe)823 6955.4 4305 2560A_(OMe)sCsC_(OMe)UC_(OMe)AC_(OMe)CA_(OMe)AG_(OMe)GC_(OMe)CA_(OMe)GC_(OMe)AsC_(OMe)sdTsdT822 − 6830.4 2531dTsdTsUsG_(OMe)GA_(OMe)GU_(OMe)GG_(OMe)UU_(OMe)CC_(OMe)GG_(OMe)UC_(OMe)GsU_(OMe)sG821 6941.3 4306 2590A_(OMe)sC_(OMe)C_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AAGGC_(OMe)C_(OMe)AGC_(OMe)AC_(OMe)dTsdT824 − 6782.4 2591dTsdTU_(OMe)GGAGU_(OMe)GGU_(OMe)U_(OMe)C_(OMe)C_(OMe)GGU_(OMe)C_(OMe)GU_(OMe)sG_(OMe)825 6893.3 4307 2620 AsCCUC_(OMe)ACC_(OMe)AAGGCC_(OMe)AGC_(OMe)ACdTsdT826 − 6698.2 2621 dTsdTUGGAGU_(OMe)GGU_(OMe)UCCGGU_(OMe)CGU_(OMe)sG 8276823.2 4308 2590A_(OMe)sC_(OMe)C_(OMe)U_(OMe)C_(OMe)AC_(OMe)C_(OMe)AAGGC_(OMe)C_(OMe)AGC_(OMe)AC_(OMe)dTsdT824 − 6782.4 2651dTsdTU_(F)GGAGU_(F)GGU_(F)U_(F)C_(F)C_(F)GGU_(F)C_(F)GU_(F)sG_(OMe) 8286785.1 4309 2620 AsCCUC_(OMe)ACC_(OMe)AAGGCC_(OMe)AGC_(OMe)ACdTsdT 826 +6698.2 2681 dTsdTUGGAGU_(F)GGU_(F)UCCGGU_(F)CGU_(F)sG 829 6775.1 4094so4326 GCACAUAGGAGAGAUGAGCUU 608 +++ ORF 343 4327 GUCGUGUAUCCUCUCUACUCGAA609 4310 2532GsC_(OMe)sAC_(OMe)AU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)sCsU_(OMe)sU832 − 7019.5 2533GsU_(OMe)sCsG_(OMe)UG_(OMe)UA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)GsA_(OMe)sA833 7487.6 4311 2562G_(OMe)sCsA_(OMe)CA_(OMe)UA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GsC_(OMe)sUsU_(OMe)834 − 7033.5 2563C_(OMe)sGsU_(OMe)sGU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)CG_(OMe)sAsA_(OMe)835 7501.7 4312 2532GsC_(OMe)sAC_(OMe)AU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)sCsU_(OMe)sU832 − 7019.5 2563C_(OMe)sGsU_(OMe)sGU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)CG_(OMe)sAsA_(OMe)835 7501.7 4313 2562G_(OMe)sCsA_(OMe)CA_(OMe)UA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GsC_(OMe)sUsU_(OMe)834 − 7033.5 2533GsU_(OMe)sCsG_(OMe)UG_(OMe)UA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)GsA_(OMe)sA833 7487.6 4314 2592GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe)sU_(OMe) 836 +6929.4 2593GsU_(OMe)C_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)C_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)AC_(OMe)U_(OMe)C_(OMe)GAsA837 7495.8 4315 2622 GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCUsU 838+++ 6887.3 2623 GsU_(OMe)CGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAsA839 7355.5 4316 2592GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe)sU_(OMe) 836+++ 6929.4 2653GsU_(F)C_(F)GU_(F)GU_(F)AU_(F)C_(F)C_(F)U_(F)C_(F)U_(F)C_(F)U_(F)AC_(F)U_(F)C_(F)GAsA840 7283.3 4317 2622 GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCUsU838 + 6887.3 2683 GsU_(F)CGU_(F)GU_(F)AU_(F)CCUCUCUAC_(F)UCGAsA 8417291.3 4107do 4117 GCACAUAGGAGAGAUGAGCdTdT 842 +++ ORF 343 4185dTdTCGUGUAUCCUCUCUACUCG 843 4318 2534GsC_(OMe)sAC_(OMe)AU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)sCsdTsdT844 + 7001.5 2535dTsdTsCsG_(OMe)UG_(OMe)UA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UsC_(OMe)sG845 6726.2 4319 2564G_(OMe)sCsA_(OMe)CA_(OMe)UA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GsC_(OMe)sdTsdT846 − 7015.5 2565dTsdTsC_(OMe)sGU_(OMe)GU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)sCsG_(OMe)847 6740.2 4320 2534GsC_(OMe)sAC_(OMe)AU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)sCsdTsdT844 − 7001.5 2565dTsdTsC_(OMe)sGU_(OMe)GU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)sCsG_(OMe)847 6740.2 4321 2564G_(OMe)sCsA_(OMe)CA_(OMe)UA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GsC_(OMe)sdTsdT846 − 7015.5 2535dTsdTsCsG_(OMe)UG_(OMe)UA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UsC_(OMe)sG845 6726.2 4322 2594G_(OMe)sC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)dTsdT 848 −6897.4 2595dTsdTC_(OMe)GU_(OMe)GU_(OMe)AU_(OMe)C_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)AC_(OMe)U_(OMe)C_(OMe)sG849 6748.3 4323 2624 GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCdTsdT850 +++ 6883.3 2625 dTsdTCGU_(OMe)GU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCsG851 6608.0 4324 2594G_(OMe)sC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)dTsdT 848 +++6897.4 2655dTsdTC_(F)GU_(F)GU_(F)AU_(F)C_(F)C_(F)U_(F)C_(F)U_(F)C_(F)U_(F)AC_(F)U_(F)C_(F)sG852 6579.9 4325 2624 GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCdTsdT850 +++ 6883.3 2685 dTsdTCGU_(F)GU_(F)AU_(F)CCUCUCUAC_(F)UCsG 853 6559.94061 4119 CAUAGGAGAGAUGAGCUUCdTdT 854 +++ ORF 346 4187dTdTGUAUCCUCUCUACUCGAAG 855 4326 2536CsA_(OMe)sUA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GC_(OMe)UU_(OMe)sCsdTsdT856 + 6939.4 2537dTsdTsGsU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)CG_(OMe)AsA_(OMe)sG857 6773.2 4327 2566C_(OMe)sAsU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)CU_(OMe)UsC_(OMe)sdTsdT858 − 6953.4 2567dTsdTsG_(OMe)sUA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)GA_(OMe)sAsG_(OMe)859 6787.3 4328 2536CsA_(OMe)sUA_(OMe)GG_(OMe)AG_(OMe)AG_(OMe)AU_(OMe)GA_(OMe)GC_(OMe)UU_(OMe)sCsdTsdT856 − 6939.4 2567dTsdTsG_(OMe)sUA_(OMe)UC_(OMe)CU_(OMe)CU_(OMe)CU_(OMe)AC_(OMe)UC_(OMe)GA_(OMe)sAsG_(OMe)859 6787.3 4329 2566C_(OMe)sAsU_(OMe)AG_(OMe)GA_(OMe)GA_(OMe)GA_(OMe)UG_(OMe)AG_(OMe)CU_(OMe)UsC_(OMe)sdTsdT858 − 6953.4 2537dTsdTsGsU_(OMe)AU_(OMe)CC_(OMe)UC_(OMe)UC_(OMe)UA_(OMe)CU_(OMe)CG_(OMe)AsA_(OMe)sG857 6773.2 4330 2596C_(OMe)sAU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe)U_(OMe)C_(OMe)dTsdT 860− 6863.4 2597dTsdTGU_(OMe)AU_(OMe)C_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)C_(OMe)U_(OMe)AC_(OMe)U_(OMe)C_(OMe)GAAsG861 6767.3 4331 2626 CsAU_(OMe)AGGAGAGAU_(OMe)GAGCUUCdTsdT 862 ++ 6807.22627 dTsdTGU_(OMe)AU_(OMe)CCUCUCUAC_(OMe)UCGAAsG 863 6641.1 4332 2596C_(OMe)sAU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe)U_(OMe)C_(OMe)dTsdT 864++ 6863.4 2657dTsdTGU_(F)AU_(F)C_(F)C_(F)U_(F)C_(F)U_(F)C_(F)U_(F)AC_(F)U_(F)C_(F)GAAsG865 6623.0 4333 2626 CsAU_(OMe)AGGAGAGAU_(OMe)GAGCUUCdTsdT 862 +++6807.2 2687 dTsdTGU_(F)AU_(F)CCUCUCUAC_(F)UCGAAsG 866 6605.0 4092 4123UGUGAAUGCAGACCAAAGAdTdT 867 +++ ORF 380 4191 dTdTACACUUACGUCUGGUUUCU 8684334 2538UsG_(OMe)sUG_(OMe)AA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)CC_(OMe)AA_(OMe)AG_(OMe)sAsdTsdT869 + 6946.5 2539dTsdTsAsC_(OMe)AC_(OMe)UU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)GG_(OMe)UU_(OMe)UsC_(OMe)sU870 6751.2 4335 2568U_(OMe)sGsU_(OMe)GA_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AC_(OMe)CA_(OMe)AA_(OMe)GsA_(OMe)sdTsdT871 − 6960.5 2569dTsdTsA_(OMe)sCA_(OMe)CU_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UG_(OMe)GU_(OMe)UU_(OMe)sCsU_(OMe)872 6765.2 4336 2538UsG_(OMe)sUG_(OMe)AA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)CC_(OMe)AA_(OMe)AG_(OMe)sAsdTsdT869 − 6946.5 2569dTsdTsA_(OMe)sCA_(OMe)CU_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UG_(OMe)GU_(OMe)UU_(OMe)sCsU_(OMe)872 6765.2 4337 2568U_(OMe)sGsU_(OMe)GA_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AC_(OMe)CA_(OMe)AA_(OMe)GsA_(OMe)sdTsdT871 + 6960.5 2539dTsdTsAsC_(OMe)AC_(OMe)UU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)GG_(OMe)UU_(OMe)UsC_(OMe)sU870 6751.2 4338 2598U_(OMe)sGU_(OMe)GAAU_(OMe)GC_(OMe)AGAC_(OMe)C_(OMe)AAAGAdTsdT 873 −6856.4 2599dTsdTACAC_(OMe)U_(OMe)U_(OMe)AC_(OMe)GU_(OMe)C_(OMe)U_(OMe)GGU_(OMe)U_(OMe)U_(OMe)C_(OMe)sU_(OMe)874 6759.3 4339 2628 UsGU_(OMe)GAAU_(OMe)GC_(OMe)AGACC_(OMe)AAAGAdTsdT875 − 6842.3 2629dTsdTAC_(OMe)AC_(OMe)UUAC_(OMe)GU_(OMe)CUGGU_(OMe)UUCsU 876 6647.1 43402598 U_(OMe)sGU_(OMe)GAAU_(OMe)GC_(OMe)AGAC_(OMe)C_(OMe)AAAGAdTsdT 873+++ 6856.4 2659dTsdTAC_(F)AC_(F)U_(F)U_(F)AC_(F)GU_(F)C_(F)U_(F)GGU_(F)U_(F)U_(F)C_(F)sU_(F)877 6586.9 4341 2628 UsGU_(OMe)GAAU_(OMe)GC_(OMe)AGACC_(OMe)AAAGAdTsdT875 ++ 6842.3 2689 dTsdTAC_(F)AC_(F)UUAC_(F)GU_(F)CUGGU_(F)UUCsU 8786586.9 4004 so 4338 GUGAAUGCAGACCAAAGAAAG 879 ++ ORF 381 4339UACACUUACGUCUGGUUUCUUUC 880 4366 2716GsU_(OMe)GA_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AC_(OMe)CA_(OMe)AA_(OMe)GA_(OMe)AA_(OMe)sG881 + 2724UsA_(OMe)CA_(OMe)CU_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UG_(OMe)GU_(OMe)UU_(OMe)CU_(OMe)UU_(OMe)sC882 4367 2717G_(OMe)sUG_(OMe)AA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)CC_(OMe)AA_(OMe)AG_(OMe)AA_(OMe)AsG_(OMe)883 − 2725U_(OMe)sAC_(OMe)AC_(OMe)UU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)GG_(OMe)UU_(OMe)UC_(OMe)W_(OMe)UsC_(OMe)884 4368 2716GsU_(OMe)GA_(OMe)AU_(OMe)GC_(OMe)AG_(OMe)AC_(OMe)CA_(OMe)AA_(OMe)GA_(OMe)AA_(OMe)sG881 + 2725U_(OMe)sAC_(OMe)AC_(OMe)UU_(OMe)AC_(OMe)GU_(OMe)CU_(OMe)GG_(OMe)UU_(OMe)UC_(OMe)UU_(OMe)UsC_(OMe)884 4369 2717G_(OMe)sUG_(OMe)AA_(OMe)UG_(OMe)CA_(OMe)GA_(OMe)CC_(OMe)AA_(OMe)AG_(OMe)AA_(OMe)AsG_(OMe)883 + 2724UsA_(OMe)CA_(OMe)CU_(OMe)UA_(OMe)CG_(OMe)UC_(OMe)UG_(OMe)GU_(OMe)UU_(OMe)CU_(OMe)UU_(OMe)sC882 4370 2715 GsU_(OMe)GAAU_(OMe)GC_(OMe)AGAC_(OMe)C_(OMe)AAAGAAAsG 885− 2723U_(OMe)sAC_(OMe)AC_(OMe)U_(OMe)U_(OMe)AC_(OMe)GU_(OMe)C_(OMe)U_(OMe)GGU_(OMe)U_(OMe)U_(OMe)C_(OMe)U_(OMe)U_(OMe)U_(OMe)sC_(OMe)886 4371 2714 GsU_(OMe)GAAU_(OMe)GC_(OMe)AGACC_(OMe)AAAGAAAsG 887 +++2722 UsACAC_(OMe)UUAC_(OMe)GU_(OMe)CUGGU_(OMe)UUCUUUsC 888 4372 2715GsU_(OMe)GAAU_(OMe)GC_(OMe)AGAC_(OMe)C_(OMe)AAAGAAAsG 885 +++ 2727U_(F)sAC_(F)AC_(F)U_(F)U_(F)AC_(F)GU_(F)C_(F)U_(F)GGU_(F)U_(F)U_(F)C_(F)U_(F)U_(F)U_(F)sC_(F)889 4373 2714 GsU_(OMe)GAAU_(OMe)GC_(OMe)AGACC_(OMe)AAAGAAAsG 887 ++2726 UsACAC_(F)UUAC_(F)GU_(F)CUGGU_(F)UUCUUUsC 890 Duplexes are shownwith the sense strand written 5′ to 3′. The complementary antisensestrand is written below the sense strand in the 3′ to 5′ direction.Lower case “d” indicates a deoxy nucleotide; all other positions areribo. Lower case “s” indicates a phosphorothioate linkage. Subscript“OMe” indicates a 2′-O-methyl sugar and subscript “F” indicates a2′-fluoro modified sugar. The extinction coefficient is the value at 260nm (*10⁻³).

TABLE 7Oligonucleotides with alternating 2′-O-methyl and 2′-fluoro modifications targeting VEGF (SEQ ID NOs 891-908), respectively Parent AL-DP- AL- AL- Effi- ObservedExtinction # DP-# SQ-# Duplex Sequence and Modifications cacy Mass OD/mgCoefficient 4060 4399 3082C_(OMe)sC_(F)C_(OMe)U_(F)G_(OMe)G_(F)U_(OMe)G_(F)G_(OMe)A_(F)C_(OMe)A_(F)U_(OMe)C_(F)− 6151.47 27.5 169 U_(OMe)U_(F)C_(OMe)C_(F)sA_(Ome) 3091G_(F)sG_(OMe)G_(F)A_(OMe)C_(F)C_(OMe)A_(F)C_(OMe)C_(F)U_(OMe)G_(F)U_(OMe)A_(F)G_(OMe)A_(F)A_(OMe)G_(F)G_(OMe)sU_(F) 4015 4400 3083G_(OMe)sG_(F)A_(OMe)C_(F)A_(OMe)U_(F)C_(OMe)U_(F)U_(OMe)C_(F)C_(OMe)A_(F)G_(OMe)G_(F) +6238.49 29 186 A_(OMe)G_(F)U_(OMe)A_(F)sC_(OMe) 3092C_(F)sC_(OMe)U_(F)G_(OMe)U_(F)A_(OMe)G_(F)A_(OMe)A_(F)G_(OMe)G_(F)U_(OMe)C_(F)C_(OMe)U_(F)C_(OMe)A_(F)U_(OMe)sG_(F) 4032 4401 3084U_(OMe)sA_(F)C_(OMe)C_(F)C_(OMe)U_(F)G_(OMe)A_(F)U_(OMe)G_(F)A_(OMe)G_(F)A_(OMe)U_(F)+++ 6239.47 31.8 188 C_(OMe)G_(F)A_(OMe)G_(F)sU_(OMe) 3093A_(F)sU_(OMe)G_(F)G_(OMe)G_(F)A_(OMe)C_(F)U_(OMe)A_(F)C_(OMe)U_(F)C_(OMe)U_(F)A_(OMe)G_(F)C_(OMe)U_(F)C_(OMe)sA_(F) 4033 4402 3085A_(OMe)sC_(F)C_(OMe)A_(F)U_(OMe)G_(F)C_(OMe)A_(F)G_(OMe)A_(F)U_(OMe)U_(F)A_(OMe)U_(F) +6262.54 30.7 194 G_(OMe)C_(F)G_(OMe)G_(F)sA_(OMe) 3094U_(F)sG_(OMe)G_(F)U_(OMe)A_(F)C_(OMe)G_(F)U_(OMe)C_(F)U_(OMe)A_(F)A_(OMe)U_(F)A_(OME)C_(F)G_(OMe)C_(F)C_(OMe)sU_(F) 4014 4403 3086G_(OMe)sC_(F)G_(OMe)G_(F)A_(OMe)U_(F)C_(OMe)A_(F)A_(OMe)A_(F)C_(OMe)C_(F)U_(OMe)C_(F)++ 6204.65 26.4 190 A_(OMe)C_(F)C_(OMe)A_(F)sA_(OMe) 3095C_(F)sG_(OMe)C_(F)C_(OMe)U_(F)A_(OMe)G_(F)U_(OMe)U_(F)U_(OMe)G_(F)G_(OMe)A_(F)G_(OMe)U_(F)G_(OMe)G_(F)UsU_(F) 4094 4404 3087G_(OMe)sC_(F)A_(OMe)C_(F)A_(OMe)U_(F)A_(OMe)G_(F)G_(OMe)A_(F)G_(OMe)A_(F)G_(OMe) +6364.57 31.3 206 so A_(F)U_(OMe)G_(F)A_(OMe)G_(F)sC_(OMe) 3096C_(F)sG_(OMe)U_(F)G_(OMe)U_(F)A_(OMe)U_(F)C_(OMe)C_(F)U_(OMe)C_(F)U_(OMe)C_(F)U_(OMe)A_(F)C_(OMe)U_(F)C_(OMe)sG_(F) 4061 4405 3088C_(OMe)sA_(F)U_(OMe)A_(F)G_(OMe)G_(F)A_(OMe)G_(F)A_(Ome)G_(F)A_(OMe)U_(F)G_(OMe)+++ 6302.59 32.8 198 A_(F)G_(OMe)C_(F)U_(OMe)U_(F)sC_(OMe) 3097G_(F)sU_(OMe)A_(F)U_(OMe)C_(F)C_(OMe)U_(F)C_(OMe)U_(F)C_(OMe)U_(F)A_(OMe)C_(F)U_(OMe)C_(F)G_(OMe)A_(F)A_(OMe)sG_(F) 4092 4406 3089U_(OMe)sG_(F)U_(OMe)G_(F)A_(OMe)A_(F)U_(OMe)G_(F)C_(OMe)A_(F)G_(OMe)A_(F)C_(OMe)++ 6309.63 33.6 207 C_(F)A_(OMe)A_(F)A_(OMe)G_(F)sA_(OMe) 3098A_(F)sC_(OMe)A_(F)C_(OMe)U_(F)U_(OMe)A_(F)C_(OMe)G_(F)U_(OMe)C_(F)U_(OMe)G_(F)G_(OMe)U_(F)U_(OMe)U_(F)C_(OMe)sU_(F) 4004 4407 3090G_(OMe)sU_(F)G_(OMe)A_(F)A_(OMe)U_(F)G_(OMe)C_(F)A_(OMe)G_(F)A_(OMe)C_(F)C_(OMe)A_(F)+++ 6332.67 30.5 213 so A_(OMe)A_(F)G_(OMe)A_(F)sA_(OMe) 3099C_(F)sA_(OMe)C_(F)U_(OMe)U_(F)A_(OMe)C_(F)G_(OMe)U_(F)C_(OMe)U_(F)G_(OMe)G_(F)U_(OMe)U_(F)U_(OMe)C_(F)U_(OMe)sU_(F) Duplexes are shown with the sense strandwritten 5′ to 3′. The complentary antisense strand is written below thesense strand in the 3′ to 5′ direction. Lower case ″s″ indicates aphosphorothioate linkage. Subscript ″OMe″ indicates a 2′-O-methyl sugarand subscript ″F″ indicates a 2′-fluoro modified sugar. The parentduplexes had unpaired nucleotides at one or both ends of the duplex.These duplexes have blunt ends. The extinction coefficient is the valueat 260 nm (*10⁻³).

TABLE 8A-BCholesterol and cholanic acid conjugates of active VEGF sequences (single strands)(SEQ ID NOs 909-922, respectively). Cal- PARENT AL-SQ culated FoundAL-DP-# # Strand SEQUENCE AND MODIFICATIONS Mass Mass Purity OD 40142363 sense GsCsGGAUCAAACCUC_(OMe)ACC_(OMe)AsAsdTsdTs- 7466.5 7463.8 98.2 Chol 4014 2697 sense Chol-sGsCGGAUC_(OMe)AAACCUC_(OMe)ACC_(OMe)Aad7232.3 7430.3  98.0 TsdT 4014 2698 senseChol-sGsCGGAUC_(OMe)AAACCUC_(OMe)AC_(OMe)C_(OMe)AAd 7446.3 7444.3  91.0TsdT 4014 2699 sense GsCGGAUC_(OMe)AAACCUC_(OMe)ACC_(OMe)AadTs-Chol7265.7 7265.7  98.0 4060 4940 senseChol-C_(OMe)C_(OMe)C_(OMe)U_(OMe)GGU_(OMe)GGAC_(OMe)AU_(OMe)C_(OMe) 100550 U_(OMe)U_(OMe)C_(OMe)C_(OMe)AdTsdT 4060 2641 senseChol-sC_(OMe)C_(OMe)C_(OMe)U_(OMe)GGU_(OMe)GGAC_(OMe)AU_(OMe)C_(OMe) 100583 U_(OMe)U_(OMe)C_(OMe)C_(OMe)AdTsdT 4033 4935 senseChol-A_(OMe)CC_(OMe)AU_(OMe)GC_(OMe)AGAU_(OMe)U_(OMe)AU_(OMe) 100 562GC_(OMe)GGAdTsdT 4033 4941 senseChol-sA_(OMe)CC_(OMe)AU_(OMe)GC_(OMe)AGAU_(OMe)U_(OMe)AU_(OMe) 100 480GC_(OMe)GGAdTsdT 4061 4936 senseChol-C_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe) 100 532U_(OMe)C_(OMe)dTsdT 4061 4942 senseChol-sC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe)U_(OMe)  98.2 514U_(OMe)C_(OMe)dTsdT 4094 2965 senseChol-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGC_(OMe) 7205.7 7205.4 89.0 U_(OMe)sU_(OMe) 4014 2701 senseGsCGGAU_(OMe)AAACCUC_(OMe)ACC_(OMe)AAdTs-Cholanic 7219.8 7219.4  88.24014 2702 sense GsCGGAUC_(OMe)AAACCUC_(OMe)AC_(OMe)C_(OMe)AAdTs-Cholanic7276.3 7274.9  71.3 4014 2696 anti-Us^(5Me)U_(F)GG^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GAUCCGCdTs- senseCholanic SEQ Cal- PARENT AL- AL- ID Effi- culated Found AL-DP-# DP-#SQ # Strand SEQUENCE AND MODIFICATIONS NO cacy MAass Mass Purity 40144206 2363 sense  GsCsGGAUCAAACCUC_(OMe)ACC_(OMe)As 909 + 7466.5 7463.898.2 AsdTsdTs-Chol 2381 as UsUGGUGAGGUUUGAUCCGCdTsdT 923 4014 4351 2697sense Chol-sGsCGGAUC_(OMe)AA ACCU 910 - 7232.3 7430.3 98.0C_(OMe)ACC_(OMe)AadTsdT 4180 as UUGGUGAGGUUUGAUCCGCTT 924 4014 4352 2698sense Chol-sGsCGGAUC_(OMe)AAACCU 911 - 7446.3 7444.3 91.0C_(OMe)AC_(OMe)C_(OMe)AAdTsdT 4180 as UUGGUGAGGUUUGAUCCGCTT 924 40144353 2699 sense GsCGGAUC_(OMe)AAACCUC_(OMe)ACC_(OMe) 912 ++ 7265.77265.7 98.0 AadTs-Chol 4180 UUGGUGAGGUUUGAUCCGCTT 924 4094 4381 2965sense  Chol-GC_(OMe)AC_(OMe)AU_(OMe)AGGAGA 919 ++ 7205.7 7205.4 89.0GAU_(OMe)GAGC_(OMe)U_(OMe)sU_(OMe) 2945 as AAGfCfUfCAfUfCfUfCfUfCfC 925fUAfUGfUGfCfUsG 4014 4209 2701 senseGsCGGAUC_(Ome)AAACCUC_(OMe)ACC_(OMe) 926 ++ 7219.8 7219.4 88.2AAdTs-Cholanic 2381 as  UsUGGUGAGGUUUGAUCCGCdTsdT 927 4014 4210 2702sense GsCGGAUC_(OMe)AAACCUC_(OMe)AC_(OMe) 928 ++ 7276.3 7274.9 71.3C_(OMe)AAdTs-Cholanic 2381 as UsUGGUGAGGUUUGAUCCGCd 923 TsdT 4014 43574112 sense GCGGAUCAAACCUCACCAATT 929 +++ 2696 anti-Us^(5Me)U_(F)GG^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F) 922 senseGAUCCGCdTs-Cholanic 4094 4390 2949 ssChol-G_(OMe)CA_(OMe)CA_(OMe)UAGGAGAG 930 +++ A_(OMe)UGAGC_(OMe)UsU 2945as AAGfCfUfCAfUfCfUfCfUfCfCf 925 UAfUGfUGfCfUsG 4094 4391 2950 ssGs_(OMe)sCA_(OMe)CA_(OMe)UAGGAGAGA_(OMe) 931 +++ UGAGC_(OMe)UU-Chol 2945as AAGfCfUfCAfUfCfUfCfUfCfCf 925 UAfUGfUGfCfUsG 4094 4392 2951 ssThio-Chol-G_(OMe)CA_(OMe)CA_(OMe)UAGG 932 +++ AGAGA_(OMe)UGAGC_(OMe)UsU2945 as AAGfCfUfCAfUfCfUfCfUfCfCf 925 UAfUGfUGfCfUsG 4094 4393 2948 ssChol-G_(OMe)CA_(OMe)CA_(OMe)UAGGAGAG 933 +++ A_(OMe)UGAGC_(OMe)UU-NH22945 as AAGfCfUfCAfUfCfUfCfUfCfCf 925 UAfUGfUGfCfUsG 4094 4394 2949 ssChol-G_(OMe)CA_(OMe)CA_(OMe)UAGGAGAG 930 +++ CA_(OMe)UGAG_(OMe)UsU 4327as AAGCUCAUCUCUCCUAUGUGCUG 934 4094 4395 2950 ssGs_(OMe)CA_(OMe)CA_(OMe)UAGGAGAGA_(OMe)U 931 +++ GAGC_(OMe)UU-Chol 4327as AAGCUCAUCUCUCCUAUGUGCUG 934 4094 4396 2951 ssThio-Chol-G_(OMe)CA_(OMe)CA_(OMe)UAGG 932 +++ AGAGA_(OMe)UGAGC_(OMe)UsU4327 as AAGCUCAUCUCUCCUAUGUGCUG 934 The strands are shown written 5′ to3′. Lower case ″s″ indicates a phosphorothioate linkage. The lower case″d″ indicates a deoxy residue. Subscript ″OMe″ indicates a 2′-O-methylsugar. Subscript ″F″ indicates a 2′-fluoro. ″Chol-″ indicates ahydroxyprolinol cholesterol conjugate. ″Cholanic″ indicates a cholanicacid conjugate. ″^(5Me)U″ indicates a 5-methyl-uridine.

TABLE 9 Naproxen conjugates of active VEGF sequence. PARENT AL- AL- AL-Effi- Calculated Found DP-# DP-# SQ # SEQUENCE AND MODIFICATIONS cacyMass Mass Purity 4014 4355 2694 asUs^(5Me)U_(F)GG^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GAUCCGCdTsdTs- +++7269.4 7270.7 80.1 Naproxen (SEQ ID NO: 935) 4112 ssGCGGAUCAAACCUCACCAATT (SEQ ID NO: 929) The antisense strand of theduplex is shown written 5′ to 3′. Lower case ″s″ indicates aphosphorothioate linkage. Lower case ″d″ indicates a deoxy. Subscript″F″ indicates a 2′-fluor sugar. ″^(5Me)U″ indicates a 5-methyl-uridine.″Naproxen″ indicates a naproxen conjugated to the oligonucleotidethrough a serinol linker.

TABLE 10 Biotin conjugates of active oligonucleotides targeting VEGF.PARENT AL- AL- Effi- Calc. Exp. AL-DP-# DP-# SQ-# StrandSequence and Modifications cacy Mass Mass Purity 4014 4356 4112 sense5 GCGGAUCAAACCUCACCAATT 3 (SEQ ID NO: 929) +++ 2695 antisUs^(5Me)U_(F)GG^(5Me)U_(F)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GAUCCGCdTsdTs-Biotin (SEQ ID NO: 936) 7285.4 7284.3 70.2 4220 3071 senseAsAGCUC_(OMe)AUCUCUCCU_(OMe)AU_(OMe)GU_(OMe)GC_(OMe)U_(OMe)sGs- Used for7872.1 7871.89 82.02 Biotin (SEQ ID NO: 937) ELISA The oligonucleotidesare written 5′ to 3′. Lower case ″s″ indicates a phosphorothioatelinkage. Lower case ″d″ indicates a deoxy. Subscript ″OMe″ indicates a2′-O-methyl sugar and subscript ″F″ indicates a 2′-fluoro modifiedsugar. ″^(5Me)U″ indicates a 5-methyl uridine.

TABLE lla-bConjugation of aldehydes, Retinal and other Retinoids to VEGF siRNAs and modeloligonucleotides. Found CGE Sequence ID Sequence* Cal Mass Mass (%)AL-3174 Q25-dTdTdTdTdTdT dTdTdTdTdTdT (SEQ ID NO: 1088)   3767.223769.09 A AL-3175 Q26-dTdTdTdTdTdT dTdTdTdTdTdT (SEQ ID NO: 1088) 3980.07 3981.37 A AL-3176Q27-dTdTdTdTdTdT dTdTdTdTdTdT (SEQ ID NO: 1088)  4034.24 4035.56 AAL-4326 GCACAUAGGAGAGAUGAGCUU (SEQ ID NO: 608) 6799.22  6798.88 AAL-3177 Q25-GCACAUAGGAGAGAUGAGCUU (SEQ ID NO: 938) B A AL-3178Q27-GCACAUAGGAGAGAUGAGCUU (SEQ ID NO: 941) 7246.66  7246.53 97% ^(C)AL-3166 GCACAUAGGAGAGAUGAGCUsU (SEQ ID NO: 1090) 6815.16  6815.10 AAL-3184 Q25-GCACAUAGGAGAGAUGAGCUsU (SEQ ID NO: 996) 6995.16  B A AL-3185Q27-GCACAUAGGAGAGAUGAGCUsU (SEQ ID NO: 1066) 7261.6 7262.47 97.8 ^(C)AL-3193 Q28-GCACAUAGGAGAGAUGAGCUsU (SEQ ID NO: 1067) 7277.61 E F AL-3211GAACUGUGUGUGAGAGGUCCsU (SEQ ID NO: 940) 6785.10 B A AL-3212Q25-GAACUGUGUGUGAGAGGUCCsU (SEQ ID NO: 1068) 6965.10 G G AL-3213Q27-GAACUGUGUGUGAGAGGUCCsU (SEQ ID NO: 1069) 7231.54 G G AL-3214Q26-GAACUGUGUGUGAGAGGUCCsU (SEQ ID NO: 1070) 7177.37 G G AL-DP-# AL-SQ-#5′-3′ Sequence Comments AL-DP-4410 AL3178Q27-GCACAUAGGAGAGAUGAGCUU (SEQ ID NO: 941) 5′Retinal4094 AL4327AAGCUCAUCUCUCCUAUGUGCUG (SEQ ID NO: 609) AL-DP-4413 AL3185Q27-GCACAUAGGAGAGAUGAGCUsU (SEQ ID NO: 1066) 5′Retinal, 3′PS 4094 AL3167AAGCUCAUCUCUCCUAUGUGCUsG (SEQ ID NO: 942) Q25 = aminooxy-linker Q26 =1-pyrene-carboxaldehyde-aminooxy Q27 = all-trans-retinal-aminooxy Q28 =4-keto-retinol A These samples were not purified and thus no CGEanalysis. B These samples were not analyzed as they were used in theconjugation reaction in the next step. ^(C) There are two isomers (E andZ) and while two peaks were seen in the CGE, only one peak was seen inthe LC/MS with one mass only. The CGE % therefore is the areas of thetwo peaks in the CGE added together. D Only a little bit of the desiredproduct was present in the crude mixture. E Two peaks in the LC/MS wereseen with masses of 7276.42 and 7277.72. The masses can be explained bythe easy oxidization of retinal to retinal. F The two main products are33% and 67% by CGE. G To be determined

TABLE 12Polyethylene glycol conjugates of active VEGF sequences and control conjugates.PARENT AL- HPLC AL- SQ MW MW retention Starting DP-# # Strand¹SEQUENCE AND MODIFICATIONS Expected Observed² time amount % Yield 40943194 VEGF GCACAUAGGAGAGAUGACGUUs-HP-NH2  7107.46 7107.2 37.497 466.67 mg 25.9 sense (SEQ ID NO: 943) 4094 3195 VEGFGCACAUAGGAGAGAUGACGUUs-HP-NH2- 27213.19  28333.51- 31.283     50 mg 33.8sense 20KPEG (SEQ ID NO: 1071) 29614.44 5167 3164 controlGsUCAUCACACUGAAUACCAAU-HP-NH2  6932.33 6932.15 19.733  491.4 mg 34.7(SEQ ID NO: 944) 5167 3170 control GsUCAUCACACUGAAUACCAAU-HP-NH2-11746.19 11000- 16.822     50 mg 38.4 5KPEG (SEQ ID NO: 1072) 13000 51673171 control GsUCAUCACACUGAAUACCAAU-HP-NH2- 26746.19 27456- 16.164    50 mg 39.2 20KPEG (SEQ ID NO: 1073) 29524 1000 2936 controlNH2-HP-CUUACGCUGAGUACUUCGAdTsdT  6915.3 6915.01 20.506 (SEQ ID NO: 945)1000 3187 control 5KPEG-NH2-HP-CUUACGCUGAGUACUUC 12021.46 11847- 17.829    50 mg 39.2 GAdTsdT (SEQ ID NO: 1074) 13256 1000 3188 control20KPEG-NH2-HP-CUUACGCUGAGUACUU 27021.46 27440- 16.921     50 mg 33.6CGAdTsdT (SEQ ID NO: 1075) 29289 1000 2937 controlCsUUACGCUGAGUACUUCGAdTdT-HP-NH2  6915.3 6915.06 20.537 (SEQ ID NO: 946)1000 3172 control CsUUACGCUGAGUACUUCGAdTdT-HP-NH2- 12021.46 12300-17.578     50 mg 48.0 5KPEG (SEQ ID NO: 1076) 13034 1000 3173 controlCsUUACGCUGAGUACUUCGAdTdT-HP-NH2- 27021.46 27000- 17.087     50 mg 52.020KPEG (SEQ ID NO: 1077) 29000 The strands are shown written 5′ to 3′.Lower case ″s″ indicates a phosphorothioate linkage. The lower case ″d″indicates a deoxy residue. ″HP-NH2″ or ″NH2-HP″ indicates ahydroxyprolinol amine conjugate used as a control. ″HP-NH2-20KPEG″ or″20KPEG-NH2-HP″ indicates conjugation to polyethylene glycol (20K)through the hydroxyprolinol linker. ″HP-NH2-5KPEG″ or ″5KPEG-NH2-HP″indicates conjugation to polyethylene glycol (5K) through thehydroxyprolinol linker. ¹The control in this column indicates that theoligonucleotide is not complementary to VEGF. Oligonucleotides 3164,3170, and 3171 target ApoB and oligonucleotides 2936, 3187, 3188, 2937,3172, and 3173 target luciferase. ²The range in observed molecularweight is due to the polydispersity of PEG starting material.

TABLE 13Oligonucleotides targeting VEGF with the ribo-difluorotoluyl modification.SEQ PARENT AL- AL- ID Duplex Sequence In vitro AL-DP-# DP-# SO-# NOsand Modifications Type efficacy T_(m)(° C.) 4014 4014 4112 806GCGGAUCAAACCUCACCAAdTdT Control +++ 80 4180 807 dTdTCGCCUAGUUUGGAGUGGUU4014 4112 806 GCGGAUCAAACCUCACCAAdTdT Mismatch + 75 2957 947dTdTCGCCUAGUUAGGAGUGGUU antisense 4014 4112 806 GCGGAUCAAACCUCACCAAdTdTMismatch + 75 2958 948 dTdTCGCCUAGUUGGGAGUGGUU antisense 4014 4112 806GCGGAUCAAACCUCACCAAdTdT Mismatch ++ 75 2959 949 dTdTCGCCUAGUUCGGAGUGGUUantisense 4014 4347 4112 806 GCGGAUCAAACCUCACCAAdTdT Difluorotoluyl ++76 2472 950 dTdTCGCCUAGUUFGGAGUGGUU 4014 4348 4112 806GCGGAUCAAACCUCACCAAdTdT Difluorotoluyl ++ 2473 951dTdTCGCCUAGUFUGGAGUGGUU 4014 4349 4112 806 GCGGAUCAAACCUCACCAAdTdTDifluorotoluyl ++ 2474 952 dTdTCGCCUAGFUFGGAGUGGUU 4014 4350 4112 806GCGGAUCAAACCUCACCAAdTdT Difluorotoluyl ′+ 70 2475 953dTdTCGCCUAGFFFGGAGUGGUU 4014 5953 954 GCGGAUCAAGCCUCACCAAdTdT Mismatch77 4180 807 dTdTCGCCUAGUUUGGAGUGGUU sense 4014 2954 955GCGGAUCAACCCUCACCAAdTdT Mismatch 73 4180 807 dTdTCGCCUAGUUUGGAGUGGUUsense 4014 2955 956 GCGGAUCAAUCCUCACCAAdTdT Mismatch 73 4180 807dTdTCGCCUAGUUUGGAGUGGUU sense Duplexes are shown with the sense strandwritten 5′ to 3′. The complementary antisense strand is written 3′ to5′. Lower case ″d″ indicates a deoxy nucleotide; all other positions areribo. Lower case ″s″ indicates a phosphorothioate linkage. ″F″ indicatesa ribo-difluorotoluyl modification. Positions altered relative to thecontrol duplex are indicated in bold face type.

TABLE 14Oligonucleotides with 2′-arafluoro-2′-deoxy-nucleosides targeting VEGF.PARENT AL- AL-sq- Effi- Expected Observed HPLC AL-DP-# DP-# # StrandSequence and Modifications cacy Mass Mass Purity 4014 4342 2478antisense UT_(araF)GGT_(araF)GAGGUUT_(araF)GAUCCGCdTdT ++ 6728.026727.25 92.82 (SEQ ID NO: 957) 4112 sense GCGGAUCAAACCUCACCAATT(SEQ ID NO: 929) 4014 4343 2479 antisenseUT_(araF)GGT_(araF)GAGGUT_(araF)T_(araF)GAUCCGCdTdT +++ 6744.04 6743.2291.97 (SEQ ID NO: 958) 4112 sense GCGGAUCAAACCUCACCAATT (SEQ ID NO: 929)4014 4344 2480 antisense UU_(araF)GGU_(araF)GAGGUUU_(araF)GAUCCGCdTdT ++6685.94 6685.13 94.83 (SEQ ID NO: 959) 4112 sense GCGGAUCAAACCUCACCAATT(SEQ ID NO: 929) 4014 4345 2481 antisenseUU_(araF)GGU_(araF)GAGGUU_(araF)U_(araF)GAUCCGCdTdT +++ 6687.93 6687.1191.97 (SEQ ID NO: 960) 4112 sense GCGGAUCAAACCUCACCAATT (SEQ ID NO: 929)4014 4346 2814 senseGCGGAUC_(araF)AA ACCUC_(araF)AC_(araF)C_(araF)AAdTdT +++ 6699.14 6698.4297.60 (SEQ ID NO: 961) 4180 antisense UUGGUGAGGUUUGAUCCGCTT(SEQ ID NO: 924) Sequences are shown written 5′ to 3′. Lower case ″d″indicates a deoxy nucleotide. ″U_(araF)″ indicates a2′-arafluoro-2′-deoxy-uridine, ″T_(araF)″ indicates a2′-arafluoro-thymidine, and ″C_(araF)″ indicates a2′-arafluoro-2′-deoxy-cytidine.

TABLE 15 Methylphosphonate-modified VEGF RNAs. PARENT AL-SQ CalculatedFound AL-DP-# # Strand SEQUENCE AND MODIFICATIONS Mass Mass Purity 40142501 sense GsCsGGAUC_(mp)AA ACCUC_(mp)A CcmpAsAsdTsdT 6712.50(SEQ ID NO: 962) 4014 2502 antisenseUsU_(mp)sGGUGAGGUU_(mp)UGAUCCGsCsdTsdT (SEQ ID NO: 963) 6758.97 6766.14014 2503 antisense UsU_(mp)sGGU_(mp)GAGGUU_(mp)U_(mp)GAUCCGsCsdTsdT6756.44 6743.99 (SEQ ID NO: 964) The oligonucleotides are shown written5′ to 3′. Lower case ″s″ indicates a phosphorothioate linkage. Subscript″mp″ indicates a methyl phosphonate linkage. Lower case ″d″ indicates adeoxy nucleotide.

TABLE 16 C-5 Allyamino-modified VEGF RNAs. PARENT AL-SQ Calculated FoundAL-DP-# # Strand SEQUENCE AND MODIFICATIONS Mass Mass Purity 4014 2504antisense UsU_(aa)sG GU_(aa)GAGGUUU_(aa)GAUCCGsCsdTsdT 6925.38 6924.992.4 (SEQ ID NO: 965) 4014 2505 antisenseUsU_(aa)sGGU_(aa)GAGGUU_(aa)U_(aa)GAUCCGsCsdTsdT 6980.40 6979.8 90.0(SEQ ID NO: 966) The oligonucleotides are shown written 5′ to 3′. Lowercase ″s″ indicates a phosphorothioate linkage. Subscript ″aa″ indicatesan allyamino modification. Lower case ″d″ indicates a deoxy nucleotide.

TABLE 17 Miscellaneous Modifications to VEGF RNA (single strands).PARENT AL-SQ SEQ ID Calculated Found AL-DP-# # Strand NOsSEQUENCE AND MODIFICATIONS Mass Mass Purity 4107 2192 sense 967GsCACAUAGGAGAGAUGAGCsdTsdT 6843.36 6842.6 84.0 4107 2193 antisense  968GsCUCAUCUCUCC*UAUGUGCsdTsdT 6584.3 6584.1 80.0 4107 2194 sense 969GsCsACAUAGGAGAGAUGAGsCsdTsdT 6875.0 6874.2 88.7 4107 2196 antisense  970GsCACAUsAGGAGAGAUGAGCsdTsdT 6875.5 6874.0 88.7 4014 2281 sense 971GsCsGGAACAAUCCUGACCAsAsdTsdT 6755.4 6753.9 82.9 mismatch 4014 2282antisense  972 UsUsGGUCAGGAUUGUUCCGsCsdTsdT 6720.0 6719.9 96.7 mismatch4014 2299 sense 973 GCGGAACAAUCCUGACCAATT 6675.0 6673.8 85.9 mismatch4014 2300 antisense  974 UUGGUCAGGAUUGUUCCGCTT 6639.9 6638.5 86.5mismatch 4014 2200 sense 975 GsCsGGAUCAAACCUCACCAsAsdTsdT 6715.4 6714.386.0 4014 2201 antisense  976 UsUsGGUGAGGUUUGAUCCGsCsdTsdT 6760.3 6759.691.2 4014 2202 sense 977 GsCGGAUCAAACCUCACCAAsdTsdT 6683.2 6682.3 95.74014 2203 antisense  978 UsUGGUGAGGUUUGAUCCGCsdTsdT 6728.1 6727.3 87.64351 2206 sense 979 UUCUUUGGUCUGCAUUCAC 5913.4 5912.3 98.0 4359 2207sense 980 UsUGGUGAGGUUUGAUCCGsCsdTsdT 6760.3 6759.05 92.0 4014 2210sense 981 GsCsGGAUCAAACCUCsACCsAsAsdTsdT 6747.5 6746.6 82.7 4014 2212sense 982 GsCsUCAUCUCUCCUsAUGUGsCsdTsdT 6616.3 6614.8 78.9 4014 2323sense 983 GsCsGGAUCAAACCUC_(OMe)ACC_(OMe)AsAsdTsdT 6743,4 6742.3 90.04014 2324 sense 984 GsCsGGAUCAAACCUOMeC_(OMe)AC_(OMe)C_(OMe)AsAsdTsdT6771.5 6770.4 86.8 4014 2325 sense 985GsCsGGAUCAAACCUC_(OMe)sACC_(OMe)sAsAsdTsdT 6775.5 6774.6 87.6 4014 2499sense 986 GsCsGGAUC_(OMe)AAACCUC_(OMe)AC_(OMe)C_(OMe)AsAsdTsdT 67716771.1 84.8 4014 2500 sense 987 GsCsGGAUdCAAACCUdCAdCdCAsAsdTsdT 6651.46650.6 82.6 4014 2506 antisense 988Us^(5Me)U_(F)sGG^(5Me)U_(F)GAGGUU^(5Me)U_(F)GAUCGsCsdTsdT 6808.4 680882.0 4014 2507 antisense 989 UsU_(F)sGG^(5Me)U_(F)GAGGU^(5Me)U_(F)^(5Me)U_(F)GAUCCGsCsdTsdT 6824.3 6823.3 80.2 4014 2508 antisense 990Us^(5Me)U_(F)sGG^(5Me)U_(F)GAGG^(5Me)U_(F) ^(5Me)U_(F)UGAUCCGsCsdTsdT6824.3 6823.4 84.3 4014 2509 antisense 991UsU_(OMe)sGGU_(OMe)GAGGU^(5Me)U_(F) ^(5Me)U_(F)GAUCCGsCsdTsdT 6820.36822.0 85.0 4220 2780 antisense 992GsC_(OMe)AC_(OMe)AU_(OMe)AGGAGAGAU_(OMe)GAGCU_(OMe)sU 6901.38 6900.778929 4060¹ 2808 sense 993 AsGsCsUsUsAsAsCsCsUsGsUsCsCsUsUsCsAsA 6230.574060¹ 2809 antisense 994 UsUsGsAsAsGsGsAsCsAsGsGsUsUsAsAsGsCsU 6413.73The oligonucleotides are shown written 5′ to 3′. Lower case ″s″indicates a phosphorothioate linkage. Lower case ″d″ indicates a deoxy.Subscript ″OMe″ indicates a 2′-O-methyl sugar. Subscript ″F″ indicates a2′-fluoro. ″^(5Me)U″ indicates a 5-methyl uridine. ¹The parent duplexhas dT overhangs. The phosphorothioate-modified duplex has blunt ends.

TABLE 18Physical characteristics of VEGF compounds derived from duplexes 4094, 4060, 4033, 4061, 4004,4014,4107 and 4003 Parent AL-SQ- SEQ ID Sense strands Calc. Obs. duplex# NOs Antisense strands mass mass AL-DP- 4326  6085′-GCACAUAGGAGAGAUGAGCUU-3′ 6670.1 6670.0 4094 4327  6093′-GUCGUGUAUCCUCUCUACUCGAA-5′ 7220.3 7220.0 Modif Seq Modifications 4554 997 5′-G*CACAUAGGAGAGAUGAGCU*U-3′ 2PS 6830.3 6830.0 4557  9985′-A*AGCUCAUCUCUCCUAUGUGCU*G-3′ 2PS 7252.4 7252.0 4555  9995′-G*CACAuAGGAGAGAUGAGCU*U-3′ 2 × PS; 1 × OMe 6844.3 6844.0 4558 10005′-A*AGCUCAUCUCUCCUAUGUGcu*G-3′ 2PS, 2 × OMe 7280.4 7280.0 4556 10015′-GcAcAuAGGAGAGAuGAGCu*U-3′ 1 × PS; 5 × OMe 6884.3 6884.0 4559 10025′-A*AGCUCAUCUCUCCuAUGUgcu*G-3 2 × PS, 3 × OMe 7294.4 7293.0 4563 10035′-G(dC)A(dC)AuAGGAGAGAuGAGCu*U-3′ 1 × PS, 3 × OMe,  6824.3 6824.0 2 ×dC 4560 1004 5′-AAGCUcAUCUCUCCuAuGuGCu*G-3′ 1 × PS, 5 × OMe 7306.47306.0 4564 1005 5′-G*CACAU_(2′F)AGGAGAGAUGAGCU*U-3′ 2 × PS; 1 × 2′F6832.2 6831.0 4561 1006 5′-AAGCUcAUCUCUCCuAuGuGcu*G-3′ 1 × PS, 6 × OMe7320.4 7320.0 4565 10075′-GC_(2F)AC_(2F)AU_(2F)AGGAGAGAU_(2F)GAGCU_(2F)*U-3′ 1 × PS; 5 × 2′F6824.3 6823.0 4562 1008 5′-AAGCU(dC)AUCUCUCCuAuGuG(dC)u*G-3′ 1 × PS, 4 ×OMe, 7260.4 7260.0 2 × dC 4566 10095′-GC_(2F)AC_(2F)AuAGGAGAGAuGAGCu*U-3′ 1 × PS, 3 × OMe, 6860.3 6859.02 × 2′F 4568 10105′-AAGCUC_(2F)AUCUCUCCU_(2F)AU_(2F)GU_(2F)GCU_(2F)*G-3′ 1 × PS, 5 × 2′F7246.4 7244.0 4567 1011 5′-GcAcAU_(2F)AGGAGAGAU_(2F)GAGCU_(2F)*U-3′ 1 ×PS, 2 × OMe, 6848.3 6847.0 3 × 2′F 4569 10125′-AAGCUcAUCUCUCCU_(2F)AU_(2F)GU_(2F)GCU_(2F)*G-3′ 1 × PS, 1 × OMe,7258.4 tbd 4 × 2′F 4567 1013 5′-GcAcAU_(2F)AGGAGAGAU_(2F)GAGCU_(2F)*U-3′1 × PS, 2 × OMe, 6848.3 6847.0 3 × 2′F 4570 10145′-AAGCUC_(2F)AUCUCUCCuAuGuGCu*G-3′ 1 × PS, 4 × OMe,  7294.4 7292.0 1 ×2′F 4571 1015 5′-GcAcAuAgGaGaGaUgAgCu*U-3′ 1 × PS, altern. 6954.3 6953.02′OMe 4572 1016 5′-aAgCuCaUcUcUcCuAuGuGcU*g-3′ 1 × PS, altern. 7404.47403.0 2′OMe 4352 1017 5′-GCACAUAGGAGAGAUGAGC-3′ blunt 6185.8 6186.04353 1018 5′-GCUCAUCUCUCCUAUGUGC-3′ blunt 5910.5 5910.8 AL-DP- 4061 10195′-CCCUGGUGGACAUCUUCCATT-3′ 6581.0 Tbd 4060 4159 10203′-TTGGGACCACCUGUAGAAGGU-5′ 6747.2 tbd Modif Seq Modifications 2580 10215′-cccuGGuGGAcAucuuccAT*T 1 × PS, 2′OMe@Py, 6765.1 6764.0 2641 10223′-T*TGGGAC_(2F)C_(2F)AC_(2F)C_(2F)U_(2F)GU_(2F)AGAAGGU_(2F)-5′ 1 ×PS, 2′F@Py 6777.3 6777.9 4934 1023 5′-(Chol)cccuGGuGGAcAucuuccAT*T 1 ×PS, 2′OMe@Py, 7470.0 7468.0 2641 10223′-T*TGGGAC_(2F)C_(2F)AC_(2F)C_(2F)U_(2F)GU_(2F)AGAAGGU_(2F)-5′ 6777.36777.9 1 × PS, 2′F@Py 4940 1024 5′-(Chol)*cccuGGuGGAcAucuuccAT*T 2 ×PS, 2′OMe@Py, 7486.0 7485.0 2641 10223′-T*TGGGAC_(2F)C_(2F)AC_(2F)C_(2F)U_(2F)GU_(2F)AGAAGGU_(2F)-5′ 5′Chol6777.3 6777.9 1 × PS, 2′F@Py AL-DP- 4026 1025 5′-ACCAUGCAGAUUAUGCGGATT6692.1 Tbd 4033 4093 1026 3′-TTUGGUACGUCUAAUACGCCU-5′ 6606.0 tbdModif Seq Modifications 2586 1027 5′-aCcAuGcAGAuuAuGcGGAT*T 1 × PS, 8 ×2′OMe 6820.2 6819.0 2647 10283′-T*TU_(2F)GGU_(2F)AC_(2F)GU_(2F)C_(2F)U_(2F)AAU_(2F)AC_(2F)GC_(2F) 1 ×PS, 2′F@Py 6644.0 6644.0 C_(2F)U_(2F) 4935 10295′-(Chol)aCcAuGcAGAuuAuGcGGAT*T 1 × PS, 8× 7525.1 Tbd 2647 10283′-T*TU_(2F)GGU_(2F)AC_(2F)GU_(2F)C_(2F)U_(2F)AAU_(2F)AC_(2F)GC_(2F)2′OMe; 5′Chol 6644.0 6644.0 C_(2F)U_(2F) 1 × PS, 2′F@Py 4941 10785′-(Chol)*aCcAuGcAGAuuAuGcGGAT*T 2 × PS, 8× 7541.1 7539.0 2647 10283′-T*TU_(2F)GGU_(2F)AC_(2F)GU_(2F)C_(2F)U_(2F)AAU_(2F)AC_(2F)GC_(2F)2′OMe, 5′Chol 6644.0 6644.0 C_(2F)U_(2F) 1 × PS, 2′F@Py AL-DP- 4119 10305′-CAUAGGAGAGAUGAGCUUCTT 6732.2 Tbd 4061 4187 10313-TTGUAUCCUCUCUACUCGAAG-5′ 6566.0 tbd Modif Seq Modifications 2596 10325′-CAuAGGAGAGAuGAGcuucT*T 1 × PS,  6846.3 6845.0 2′OMe@allPy  2657 10333′-TTGuAuccucucuACucGAAG-5′ 1 × PS, 2′F@Py 6604.1 6605.0 4936 10345′-(Chol)CAuAGGAGAGAuGAGcuucT*T 1 × PS, 2′OMe@Py, 7551.2 Tbd 2657 10353′-TTGuAuccucucuACucGAAG-5′ 5′Chol 6604.1 6605.0 1 × PS, 2′F@Py 49371079 5′-(Chol)*CAuAGGAGAGAuGAGcuucT*T 2 × PS, 2′OMe@Py, 7567.2 7565.02657 1035 3′-TTGuAuccucucuACucGAAG-5′ 5′Chol 6604.1 6605.0 1 ×PS, 2′F@Py AL-DP- 2626 1036 5′-cAuAGGAGAGAuGAGCUUCT*T-3′ 1 × PS, 3 ×2′OMe 6790.3 6789.0 4331 2627 1037 3′-T*TGuAuCCUCUCUAcUCGAAG-5′ 1 ×PS, 3 × 2′OMe 6624.1 6624.0 AL-DP- 4338 1038 5′-GUGAAUGCAGACCAAAGAAAG-3′6828.3 tbd 4004 4339 1039 3′-UACACUUACGUCUGGUUUCUUUC-5′ Modif SeqModifications 4350 1040 5′-GUGAAUGCAGACCAAAGAA-3′ blunt 6153.8 6154.04351 1041 5′-UUCUUUGGUCUGCAUUCAC-3′ blunt 5912.5 5911.8 4338 10385′-GUGAAUGCAGACCAAAGAAAG3′ blunt 6829.3 6523.5 4344 10425′-CUUUCUUUGGUCUGCAUUCAC-3′ blunt 6523.9 AL-DP- 2714 10435′-GuGAAuGcAGACcAAAGAAA*G-3′ 1 × PS, 4 × 2′OMe 6900.4 6900.0 4371 27221044 3′-U*ACAcUUAcGuCUGGuUUCUUUC-5′ 1 × PS, 4 × 2′OMe 7231.3 7230.0AL-DP- 4112 806 5′-GCGGAUCAAACCUCACCAATT-3′ 6634.1 6634.5 4014 4180 8073′-TTCGCCUAGUUUGGAGUGGUU-5′ 6679.1 6680.3 Modif Seq Modifications 43181045 5′-GCGGAUCAAACCUCACCAAGG-3′ blunt 6717.2 tbd 4342 10465′-CCUUGGUGAGGUUUGAUCCGC-3′ blunt 6681.0 6683.3 4346 10475′-GCGGAUCAAACCUCACCAA-3′ blunt 6025.7 6026.5 4347 10485′-UUGGUGAGGUUUGAUCCGC-3′ blunt 6070.6 6071.3 AL-DP- 4358 10495′-G*C*GGAUCAAACCUCACCA*A*T*T-3′ (2 + 3)PS 6714.4 6714.8 4127 2201 10503′-T*T*C*GCCUAGUUUGGAGUGG*U*U-5′ (2 + 3)PS 6759.3 tbd AL-DP- 4117 10515′-GCACAUAGGAGAGAUGAGCTT-3′ 6794.2 6794.0 4107 4185 10523′-TTCGUGUAUCCUCUCUACUCG-5′ 6518.9 6519.0 Modif Seq Modifications 43261053 5′-GCACAUAGGAGAGAUGAGCUU-3′ 6799.2 tbd 4345 10545′-AAGCUCAUCUCUCCUAUGUGC-3′ blunt 6569.0 6568.5 4354 10555′-G*CACAUAGGAGAGAUGAGC*T*T-3′ (1 + 2)PS 6842.4 6842.5 4356 10565′-G*C*ACAUAGGAGAGAUGAG*C*T*T-3′ (2 + 3)PS 6874.5 tbd AL-DP- 4286 10575′-GGACAUCUUCCAGGAGUACCC-3′ 6670.1 6669.5 4003 4287 10585′-GGGUACUCCUGGAAGAUGUCCAC-3′ 7361.5 7362.0 Modif Seq Modifications 43481059 5′-GGACAUCUUCCAGGAGUAC-3′ Blunt 6059.7 6059.5 4349 10605′-GUACUCCUGGAAGAUGUCC-3′ blunt 6036.7 6036.8 4286 10575′-GGACAUCUUCCAGGAGUACCC-3′ blunt 6671.1 tbd 4343 10615′-GGGUACUCCUGGAAGAUGUCC-3′ blunt 6727.1 6727.5 Abbreviations used:Lower case letters: 2′OMe ribonucleotides T: Deoxythymidine (Chol):Cholesterol Upper case letters followed by subscript 2′F: 2′Fribonucleotides (dC): Deoxycytidine Tbd: to be determined Upper caseletters: regular ribonucleotides *: Phosphorothioate linkage Altern.:alternating

1. An isolated double stranded iRNA agent, wherein the iRNA agentcomprises a sense strand and an antisense strand, wherein the antisensestrand comprises a sequence complementary to 19- to 23 nucleotides of aVEGF nucleotide sequence and each strand is 15-30 nucleotides in length.2. The iRNA agent of claim 1, wherein the sense sequence comprises asequence that differs by no more than 1, 2, or 3 nucleotides from asequence selected from the group consisting of SEQ ID NOs:2-401 and SEQID NO:456, SEQ ID NO:546, SEQ ID NO:548, SEQ ID NO:550, SEQ ID NO:552,SEQ ID NO:590, SEQ ID NO:592, SEQ ID NO:594, SEQ ID NO:596, SEQ IDNO:608, SEQ ID NO:610, SEQ ID NO:612, SEQ ID NO:614, SEQ ID NO:634, SEQID NO:636, SEQ ID NO:638, SEQ ID NO:640, SEQ ID NO:646, SEQ ID NO: 648,SEQ ID NO:650 and SEQ ID NO:671.
 3. The iRNA agent of claim 1, whereineach strand is 19-23 nucleotides in length.
 4. The iRNA agent of claim1, wherein at least one strand comprises at least one nucleotideoverhang having 1 to 4 nucleotides.
 5. The iRNA agent of claim 1,wherein the iRNA agent comprises at least one non-nucleotide moiety. 6.The iRNA agent of claim 1, wherein at least one nucleotide of at leastone strand of the iRNA agent is a 2′-modified nucleotide comprising amodification selected from the group consisting of: 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-β-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).
 7. The iRNA agentof claim 1, wherein the iRNA agent comprises a cholesterol moiety. 8.The iRNA agent of claim 1, wherein the iRNA agent comprises acholesterol moiety conjugated to the 3′-end of the sense strand of theiRNA agent and/or to the 3′-end of the antisense strand of the iRNAagent.
 9. A cell comprising the iRNA agent of claim
 1. 10. Apharmaceutical composition comprising the iRNA agent of claim 1 and apharmaceutically acceptable carrier.
 11. A method of inhibiting VEGFexpression in a cell comprising contacting the cell with the iRNA agentof claim 1 and maintain the cell for a time sufficient to obtaindegradation of the mRNA transcript of a VEGF gene, thereby inhibitingexpression of VEGF in the cell.
 12. The method of claim 11, wherein thecell is a human cell of a human subject.
 13. The method of claim 12,wherein the subject is diagnosed as having an ophthalmic disorder ormacular degeneration or diabetic retinopathy or cancer.
 14. The methodof claim 11, wherein contacting the cell with the iRNA agent results inat least 80% inhibition of expression of an endogenous human VEGF121gene in HeLA cells measured by ELISA assay or results in at least 95%inhibition of expression of an endogenous human VEGF121 gene in HeLAcells under hypoxic conditions as measured by ELISA assay or results ingreater than 90% inhibition of expression of an endogenous human VEGF121gene in HeLA cells as measured by ELISA assay, and wherein the iRNAagent is modified with a phosphorothioate linkage, a 2′-O-methylnucleotide, or a 2′-fluoro-modified nucleotide.
 15. A method of treatinga disease or condition associated with VEGF expression in a subjectcomprising administration of an effective amount of the iRNA agent ofclaim 1 to the subject.
 16. The method of claim 15, wherein disease orcondition comprises adult onset macular degeneration, diabeticretinopathy, neovascular glaucoma, colon cancer, breast cancer, renalcancer, pulmonary disease, rheumatoid arthritis, or psoriasis.
 17. Themethod of claim 15, wherein administration is at or near the site ofVEGF expression.
 18. The method of claim 15, wherein administration isto a cell or cells in a choroid region of the eye by injection.
 19. Themethod of claim 18, wherein administration is performed at a unit doseselected from a group consisting of: about 0.00001 mg to about 3 mg pereye; about 0.0001-0.001 mg per eye; about 0.03-3.0 mg per eye; about0.1-3.0 mg per eye; and about 0.3-3.0 mg per eye.
 20. The method ofclaim 15, comprising multiple administrations.